Insoluble insulin compositions

ABSTRACT

The present invention relates to insoluble compositions containing acylated proteins selected from the group consisting of acylated insulin, acylated insulin analog, and acylated proinsulin, and formulations thereof. The formulations are suitable for parenteral delivery or other means of delivery, to a patient for extended control of blood glucose levels. More particularly, the present invention relates to compositions comprised of an acylated protein complexed with zinc, protamine, and a phenolic compound such that the resulting microcrystal is analogous to the neutral protamine Hagedorn (NPH) insulin crystal form. Surprisingly, it has been discovered that compositions of such acylated proteins have therapeutically superior subcutaneous release pharmacokinetics, and more extended and flatter glucodynamics, than presently available commercial preparations of NPH insulin. Yet, the present crystals retain certain advantageous properties of NPH crystals, being readily able to be resuspended and also mixable with soluble insulins.

[0001] This application claims priority to U.S. Provisional ApplicationSer. No. 60/063104, filed on Oct. 24, 1997, and U.S. ProvisionalApplication Ser. No. 60/088930, filed Jun. 11, 1998.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention is in the field of human medicine. Moreparticularly, this invention is in the field of pharmaceutical treatmentof the diseases of diabetes and hyperglycemia.

[0004] 2. Description of Related Art

[0005] It has long been a goal of insulin theraphy of mimic the patternof endogenous insulin secretion in normal individuals. The dailyphysiological deman for insulin fluctuates and can be separated into twophases: (a) the absorptive phase requiring a pulse of insulin to disposeof the meal-related blood glucose surge, and (b) the post-absorptivephase requiring a sustained delivery of insulin to regulate hepaticglucose output for maintaining optimal fasting blood glucose.

[0006] Accordingly, effective therapy for people with diabetes generallyinvolves the combined use of two types of exogenous insulinformulations: a rapid acting meal time insulin provided by bolusinjections and a long-acting, so-called, basal insulin, administered byinjection once or twice daily to control blook glucose levels betweenmeals. An ideal basal insulin will provide an extended and “flat” timeaction—that is, it will control blood glucose levels for at least 12hours, and preferably for 24 hours or more, without significant risk ofhypoglycemia. Furthermore, an ideal basal insulin should be mixable witha soluble meal-time insulin, and should not cause irritation or reactionat the site of administration. Finally, basal insulin preparations thatare suspension formulations should be able to be readily, and uniformlyresuspended by the patient prior to administration.

[0007] As is well understood by those skilled in this art, long-actinginsulin formulations have been obtained by formulating normal insulin asmicrocrystalline suspensions for subcutaneous injection. Examples ofcommercial basal insulin preparations include NPH (Neutral ProtamineHagedorn) insulin, protamine zinc insulin (PZI), and ultralente (UL).NPH insulin is the most widely-used insulin preparation, constitutingfrom 50 to 70 per cent of the insulin used worldwide. It is a suspensionof a microcrystalline complex of insulin, zinc, protamine, and one ormore phenolic preservatives. NPH insulin preparations are commerciallyavailable incorporating human insulin, pork insulin, beef insulin, ormixtures thereof. Also, NPH-like preparations of a monomeric insulinanalog, LysB298,ProB29-human insulin analog, are known in the art[abbreviated herein as “NPL”: De Felippis, M. R., U.S. Pat. No.5,461,031, issued Oct. 24, 1995; De Felippis, M. R., U.S. Pat. No.5,650,486, issued Jul. 22, 1997; and De Felippis, M. R., U.S. Pat. No.5,747,642, issued May 5, 1998].

[0008] NPH insulin microcrystals possess a distinctive rod-shapedmorphology of typical dimensions about 5 microns long by 1 micron thickand 1 micron wide. The extended duration of action of NPH insulinmicrocrystals results from their slow absorption from the subcutaneousinjection site.

[0009] Therapy using currently-available NPH insulin preparations failsto provide the ideal “flat” pharmacokinetics necessary to maintainoptimal fasting blood glucose for an extended period of time betweenmeals. Consequently, treatment with NPH insulin can result inundesirably high levels of insulin in the blood, which may causelife-threatening hypoglycemia.

[0010] In addition to failing to provide an ideal flat pharmacokineticprofile, the duration of action of NPH insulin also is not ideal. Inparticular, a major problem with NPH therapy is the “dawn phenomenon”which is hyperglycemia that results from the loss of effective glucosecontrol overnight while the patient is sleeping. These deficiencies inglycemic control contribute to serious long-term medical complicationsof diabetes and impose considerable inconvenience and quality-of-lifedisadvantages to the patient.

[0011] Protamine zinc insulin (PZI) has a composition similar to NPH,but contains higher levels of protamine and zinc than NPH. PZIpreparations may be made as intermediate-acting amorphous precipitatesor long-acting crystalline material. PZI, however, is not an ideal basalinsulin pharmaceutical because it is not mixable with a solublemeal-time insulin, and the high zinc and protamine can cause irritationor reaction at the site of administration.

[0012] Human insulin ultralente is a microcrystalline preparation ofinsulin having higher levels of zinc than NPH, and not having eitherprotamine or a phenolic preservative incorporated into the microcrystal.Human ultralente preparations provide moderate time action that is notsuitably flat, and they do not form stable mixtures with insulin.Furthermore, they are difficult to resuspend.

[0013] There have been attempts to address the perceived inadequacies ofknown insulin suspensions. Fatty acid-acylated insulins have beeninvestigated for basal control of blood glucose [Havelund, S., et al.,WIPO publication WO95/07931, Mar. 23, 1995]. Their extended time actionis caused by binding of the fatty acyl portion of these molecules toserum albumin. The fatty acyl chain lengths of these molecules is suchas to take advantage of the fatty acid binding capability of serumalbumin. The fatty acid chains used in fatty acid-acylated insulins aretypically longer than about ten carbon atoms, and chain lengths offourteen and sixteen carbon atoms are optimal for binding to serumalbumin and extending time action.

[0014] Unlike NPH insulin, which is insoluble, the aforementioned fattyacid-acylated insulins are soluble at the usual therapeuticconcentrations of insulin. However, the time action of thesepreparations may not be sufficiently long enough, or flat enough, toprovide ideal basal control, and they are less potent than insulin,thereby requiring administration of greater amounts of the drug agent[Radziuk, J., et al., Diabetologia 41:116-120, 489-490 (1998)].

[0015] Whittingham, J. L., et al. [Biochemistry 36:2826-2831 (1997)]crystallized B29-Nε-tetradecanoyl-des(B30)-human insulin analog as ahexamer complex with zinc and phenol for the purpose of structuralstudies by X-ray crystallography. The hexamer was found to be in the R6conformation, and to have certain properties different from hexamers ofhuman insulin. Whittingham, et al. do not disclose any pharmaceutical orpharmacological properties of the crystal that was formed, nor do theysuggest that such a crystal would have any advantageous properties fortreating diabetes or hyperglycemia. It is not possible to predict fromWhittingham, et al. whether protamine-containing crystals of the NPHtype could be formed with derivatized insulins and insulin analogs, orwhat the pharmacokinetics or pharmacodynamic response of such crystalswould be.

[0016] Thus, there remains a need to identify insulin preparations thathave flatter and longer time action than NPH insulin, that are mixablewith soluble, meal-time insulins, that can be readily resuspended, andthat do not pose risk of irritation or reaction at the site ofadministration.

SUMMARY OF THE INVENTION

[0017] I have unexpectedly observed that when insulin is made lesssoluble by derivatizing one or more of its reactive side groups, thederivatized insulin can be incorporated into NPH-like crystals withprotamine. When the derivatized protein is precipitated or crystallized,the rate at which the insulin derivative dissolves from the solid formis greatly reduced compared with the rate at which similar solid formscomprised of un-derivatized protein dissolve. I have furthermorediscovered that crystals of derivatized proteins provide flatter andlonger time action than do crystals comprised of un-derivatized protein.Additionally, I have surprisingly discovered that the benefits offlatter and longer time action can be obtained even from amorphousprecipitates comprising derivatized protein.

[0018] Accordingly, in its broadest aspect, the present inventionprovides insoluble compositions comprising a derivatized proteinselected from the group consisting of insulin derivatives, insulinanalog derivatives, and proinsulin derivatives, wherein the derivativesare less soluble than the underivatized insulin, insulin analog, orproinsulin. The insoluble compositions also are comprised of acomplexing compound, a hexamer-stabilizing compound, and a divalentmetal cation. These insoluble compositions are useful for treatingdiabetes and hyperglycemia, and provide the advantages of having flatterand longer time action than NPH insulin. Furthermore, they are mixablein a formulation with soluble protein and with soluble derivatizedprotein. The insoluble compositions of the present invention are in theform of amorphous precipitates, and also more preferably, in the form ofmicrocrystals.

[0019] More specifically, the present invention providesmicrocrystalline forms of fatty acid-acylated proteins that are usefulfor treating diabetes and hyperglycemia. These microcrystals comprise afatty acid-acylated protein selected from the group consisting of fattyacid-acylated insulin, fatty acid-acylated insulin analog, and fattyacid-acylated proinsulin, protamine, a phenolic preservative, and zinc.Such microcrystals will provide both flatter and longer time action thanNPH insulin, and are mixable with soluble proteins and solublederivatized proteins.

[0020] The invention provides aqueous suspension formulations comprisingthe insoluble composition and an aqueous solvent. Such suspensionformulations may contain, optionally, a soluble protein, such as humaninsulin, or a soluble analog of human insulin, such as a monomericinsulin analog, that control blood glucose immediately following a meal.The microcrystalline formulations of fatty acid-acylated insulins havesuperior pharmacodynamics compared with human insulin NPH. The presentinvention is distinct from previous fatty acid-acylated insulintechnology in that the extension of time action of the present inventiondoes not rely necessarily on albumin-binding, though albumin binding mayfurther protract the time action of certain of the compositions of thepresent invention.

[0021] The invention also pertains to a process for preparing theinsoluble compositions, and a method of treating diabetes orhyperglycemia comprising administering a formulation containing aninsoluble composition to a patient in need thereof in a quantitysufficient to regulate blood glucose levels in the patient.

[0022] Also part of the present invention are amorphous precipitates,comprising, in their broadest aspect, a derivatized protein selectedfrom the group consisting of derivatized insulin, derivatized insulinanalog, and derivatized proinsulin, protamine, a phenolic preservative,and zinc, wherein the derivatized protein is less soluble than theunderivatized protein.

BRIEF DESCRIPTION OF THE DRAWING

[0023] The dissolution rate of pork insulin NPH (- - -) and ofB29-Nε-octanoyl-human insulin microcrystals of this invention (——————)are compared in FIG. 1.

DESCRIPTION OF THE INVENTION

[0024] The term “insoluble composition” refers to matter in either amicrocrystalline state or in an amorphous precipitate state. Thepresence of microcrystals or amorphous precipitate can be ascertained byvisual and microscopic examination. Solubility depends on solvent, and aparticular composition may be insoluble in one solvent, but soluble inanother.

[0025] The term “microcrystal” means a solid that is comprised primarilyof matter in a crystalline state, wherein the individual crystals arepredominantly of a single crystallographic composition and are of amicroscopic size, typically of longest dimension within the range 1micron to 100 microns. The term “microcrystalline” refers to the stateof being a microcrystal.

[0026] The term “amorphous precipitate” refers to insoluble materialthat is not crystalline in form. The person of ordinary skill candistinguish crystals from amorphous precipitate. The amorphousprecipitates of the present invention have advantageous pharmacologicalproperties in their own right, and also are intermediates in theformation of the microcrystals of the present invention.

[0027] The term “derivatized protein” refers to a protein selected fromthe group consisting of derivatized insulin, derivatized insulinanalogs, and derivatized proinsulin that is derivatized by a functionalgroup such that the derivatized protein is less soluble in an aqueoussolvent than is the un-derivatized protein. Many examples of suchderivatized proteins are known in the art, and the determination ofsolubility of proteins and derivatized proteins is well-known to theskilled person. Examples of derivatized insulin and insulin analogsinclude benzoyl, p-tolyl-sulfonamide carbonyl, and indolyl derivativesof insulin and insulin analogs [Havelund, S., et al., WO95/07931,published Mar. 23, 1995]; alkyloxycarbonyl derivatives of insulin[Geiger, R., et al., U.S. Pat. No. 3,684,791, issued Aug. 15, 1972;Brandenberg, D., et al., U.S. Pat. No. 3,907,763, issued Sep. 23, 1975];aryloxycarbonyl derivatives of insulin [Brandenberg, D., et al., U.S.Pat. No. 3,907,763, issued Sep. 23, 1975]; alkylcarbamyl derivatives[Smyth, D. G., U.S. Pat. No. 3,864,325, issued Feb. 4, 1975; Lindsay, D.G., et al., U.S. Pat. No. 3,950,517, issued Apr. 13, 1976]; carbamyl,O-acetyl derivatives of insulin [Smyth, D. G., U.S. Pat. No. 3,864,325issued Feb. 4, 1975]; cross-linked, alkyl dicarboxyl derivatives[Brandenberg, D., et al., U.S. Pat. No. 3,907,763, issued Sep. 23,1975]; N-carbamyl, O-acetylated insulin derivatives [Smyth, D. G., U.S.Pat. No. 3,868,356, issued Feb. 25, 1975]; various O-alkyl esters[Markussen, J., U.S. Pat. No. 4,343,898, issued Aug. 10, 1982; Morihara,K., et al., U.S. Pat. No. 4,400,465, issued Aug. 23, 1983; Morihara, K.,et al., U.S. Pat. No. 4,401,757, issued Aug. 30, 1983; Markussen, J.,U.S. Pat. No. 4,489,159, issued Dec. 18, 1984; Obermeier, R., et al.,U.S. Pat. No. 4,601,852, issued Jul. 22, 1986; and Andresen, F. H., etal., U.S. Pat. No. 4,601,979, issued Jul. 22, 1986]; alkylamidederivatives of insulin [Balschmidt, P., et al., U.S. Pat. No. 5,430,016,issued Jul. 4, 1995]; various other derivatives of insulin [Lindsay, D.G., U.S. Pat. No. 3,869,437, issued Mar. 4, 1975]; and the fattyacid-acylated proteins that are described herein.

[0028] The term “acylated protein” as used herein refers to aderivatized protein selected from the group consisting of insulin,insulin analogs, and proinsulin that is acylated with an organic acidmoiety that is bonded to the protein through an amide bond formedbetween the acid group of an organic acid compound and an amino group ofthe protein. In general, the amino group may be the a-amino group of anN-terminal amino acid of the protein, or may be the ε-amino group of aLys residue of the protein. An acylated protein may be acylated at oneor more of the three amino groups that are present in insulin and inmost insulin analogs. Mono-acylated proteins are acylated at a singleamino group. Di-acylated proteins are acylated at two amino groups.Tri-acylated proteins are acylated at three amino groups. The organicacid compound may be, for example, a fatty acid, an aromatic acid, orany other organic compound having a carboxylic acid group that will forman amide bond with an amino group of a protein, and that will cause theaqueous solubility of the derivatized protein to be lower than thesolubility of the un-derivatized protein.

[0029] The term “fatty acid-acylated protein” refers to a an acylatedprotein selected from the group consisting of insulin, insulin analogs,and proinsulins that is acylated with a fatty acid that is bonded to theprotein through an amide bond formed between the acid group of the fattyacid and an amino group of the protein. In general, the amino group maybe the α-amino group of an N-terminal amino acid of the protein, or maybe the ε-amino group of a Lys residue of the protein. A fattyacid-acylated protein may be acylated at one or more of the three aminogroups that are present in insulin and in most insulin analogs.Mono-acylated proteins are acylated at a single amino group. Di-acylatedproteins are acylated at two amino groups. Tri-acylated proteins areacylated at three amino groups. Fatty acid-acylated insulin is disclosedin a Japanese patent application 1-254,699. See also, Hashimoto, M., etal., Pharmaceutical Research, 6:171-176 (1989), and Lindsay, D. G., etal., Biochemical J. 121:737-745 (1971). Further disclosure of fattyacid-acylated insulins and fatty acylated insulin analogs, and ofmethods for their synthesis, is found in Baker, J. C., et al, U.S. Ser.No. 08/342,931, filed Nov. 17, 1994 and issued as U.S. Pat. No.5,693,609, Dec. 2, 1997; Havelund, S., et al., WO95/07931, publishedMar. 23, 1995, and a corresponding U.S. Pat. No. 5,750,497, May 12,1998; and Jonassen, I., et al., WO96/29342, published Sep. 26, 1996.These disclosures are expressly incorporated herein by reference fordescribing fatty acid-acylated insulins and fatty acid-acylated insulinanalogs and for enabling preparation of the same.

[0030] The term “fatty acid-acylated protein” includes pharmaceuticallyacceptable salts and complexes of fatty acid-acylated proteins. The term“fatty acid-acylated protein” also includes preparations of acylatedproteins wherein the population of acylated protein molecules ishomogeneous with respect to the site or sites of acylation. For example,Nε-mono-acylated protein, B1-Nα-mono-acylated protein,A1-Nα-mono-acylated protein, A1, B1-Nα-di-acylated protein, Nε,A1-Nα,di-acylated protein, Nε,B1-Nα,di-acylated protein, andNε,A1,B1-Nα,tri-acylated protein are all encompassed within the term“fatty acid-acylated protein” for the purpose of the present invention.The term also refers to preparations wherein the population of acylatedprotein molecules has heterogeneous acylation. In the latter case, theterm “fatty acid-acylated protein” includes mixtures of mono-acylatedand di-acylated proteins, mixtures of mono-acylated and tri-acylatedproteins, mixtures of di-acylated and tri-acylated proteins, andmixtures of mono-acylated, di-acylated, and tri-acylated proteins.

[0031] The term “insulin” as used herein, refers to human insulin, whoseamino acid sequence and special structure are well-known. Human insulinis comprised of a twenty-one amino acid A-chain and a thirty-amino acidB-chain which are cross-linked by disulfide bonds. A properlycross-linked insulin contains three disulfide bridges: one betweenposition 7 of the A-chain and position 7 of the B-chain, a secondbetween position 20 of the A-chain and position 19 of the B-chain, and athird between positions 6 and 11 of the A-chain.

[0032] The term “insulin analog” means proteins that have an A-chain anda B-chain that have substantially the same amino acid sequences as theA-chain and B-chain of human insulin, respectively, but differ from theA-chain and B-chain of human insulin by having one or more amino aciddeletions, one or more amino acid replacements, and/or one or more aminoacid additions that do not destroy the insulin activity of the insulinanalog.

[0033] “Animal insulins” are insulin analogs. Four such animal insulinsare rabbit, pork, beef, and sheep insulin. The amino acid substitutionsthat distinguish these animal insulins from human insulin are presentedbelow for the reader's convenience. Amino Acid Position A8 A9 A10 B30human insulin Thr Ser Ile Thr rabbit insulin Thr Ser Ile Ser porkinsulin Thr Ser Ile Ala beef insulin Ala Ser Val Ala sheep insulin AlaGly Val Ala

[0034] Another type of insulin analog, “monomeric insulin analog” iswell-known in the art. Monomeric insulin analogs are structurally verysimilar to human insulin, and have activity similar or equal to humaninsulin, but have one or more amino acid deletions, replacements oradditions that tend to disrupt the contacts involved in dimerization andhexamerization which results in their greater tendency to dissociate toless aggregated states. Monomeric insulin analogs are rapid-actinganalogs of human insulin, and are disclosed, for example, in Chance, R.E., et al., U.S. Pat. No. 5,514,646, May 7, 1996; Brems, D. N., et al.Protein Engineering, 5:527-533 (1992); Brange, J. J. V., et al., EPOpublication No. 214,826, published Mar. 18, 1987; Brange, J. J. V., etal., U.S. Pat. No. 5,618,913, Apr. 8, 1997; and Brange, J., et al.,Current Opinion in Structural Biology 1:934-940 (1991). An example ofmonomeric insulin analogs is described as human insulin wherein Pro atposition B28 is substituted with Asp, Lys, Leu, Val, or Ala, and whereinLys at position B29 is Lys or is substituted with Pro, and also,AlaB26-human insulin, des(B28-B30)-human insulin, and des(B27)-humaninsulin. The monomeric insulin analogs employed as derivatives in thepresent crystals, or employed un-derivatized in the solution phase ofsuspension formulations, are properly cross-linked at the same positionsas is human insulin.

[0035] Another group of insulin analogs for use in the present inventionare those wherein the isoelectric point of the insulin analog is betweenabout 7.0 and about 8.0. These analogs are referred to as “pI-shiftedinsulin analogs.” Examples of such insulin analogs includeArgB31,ArgB32-human insulin, GlyA21,ArgB31,ArgB32-human insulin,ArgA0,ArgB31,ArgB32-human insulin, and ArgA0,GlyA21,ArgB31,ArgB32-humaninsulin.

[0036] Another group of insulin analogs consists of insulin analogs thathave one or more amino acid deletions that do not significantly disruptthe activity of the molecule. This group of insulin analogs isdesignated herein as “deletion analogs.” For example, insulin analogswith deletion of one or more amino acids at positions B1-B3 are active.Likewise, insulin analogs with deletion of one or more amino acids atpositions B28-B30 are active. Examples of “deletion analogs” includedes(B30)-human insulin, desPhe(B1)-human insulin, des(B27)-humaninsulin, des(B28-B30)-human insulin, and des(B1-B3)-human insulin. Thedeletion analogs employed as derivatives in the present crystals, oremployed un-derivatized in the solution phase of suspensionformulations, are properly cross-linked at the same positions as ishuman insulin.

[0037] Optionally, an insulin analog may have replacements of one ormore of its amidated amino acids with other amino acids for the sake ofchemical stability. For example, Asn and Gln may be replaced with Gly,Ser, Thr, Asp or Glu. In particular, AsnA18, AsnA21, or AsnB3, or anycombination of those residues may be replaced by Gly, Asp, or Glu, forexample. Also, GlnA15 or GlnB4, or both, may be replaced by either Aspor Glu. Preferred replacements are Asp at B21, and Asp at B3.

[0038] The term “proinsulin” means a single-chain peptide molecule thatis a precursor of insulin. Proinsulin may be converted to insulin or toan insulin analog by chemical or, preferably, enzyme-catalyzedreactions. In proinsulin, proper disulfide bonds are formed as describedherein. Proinsulin comprises insulin or an insulin analog and aconnecting bond or a connecting peptide. A connecting peptide hasbetween 1 and about 35 amino acids. The connecting bond or connectingpeptide connects to a terminal amino acid of the A-chain and to aterminal amino acid of the B-chain by an α-amide bond or by two α-amidebonds, respectively. Preferably, none of the amino acids in theconnecting peptide is cysteine. Preferably, the C-terminal amino acid ofthe connecting peptide is Lys or Arg. Proinsulin may have the formulaX-B-C-A-Y or may have the formula X-A-C-B-Y, wherein X is hydrogen or isa peptide of from 1 to about 100 amino acids that has either Lys or Argat its C-terminal amino acid, Y is hydroxy, or is a peptide of from 1 toabout 100 amino acids that has either Lys or Arg at its N-terminal aminoacid, A is the A-chain of insulin or the A-chain of an insulin analog, Cis a peptide of from 1 to about 35 amino acids, none of which iscysteine, wherein the C-terminal amino acid is Lys or Arg, and B is theB-chain of insulin or the B-chain of an insulin analog.

[0039] A “pharmaceutically acceptable salt” means a salt formed betweenany one or more of the charged groups in a protein and any one or morepharmaceutically acceptable, non-toxic cations or anions. Organic andinorganic salts include, for example, those prepared from acids such ashydrochloric, sulfuric, sulfonic, tartaric, fumaric, hydrobromic,glycolic, citric, maleic, phosphoric, succinic, acetic, nitric, benzoic,ascorbic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic,propionic, carbonic, and the like, or for example, ammonium, sodium,potassium, calcium, or magnesium.

[0040] The verb “acylate” means to form the amide bond between a fattyacid and an amino group of a protein. A protein is “acylated” when oneor more of its amino groups is combined in an amide bond with the acidgroup of a fatty acid.

[0041] The term “fatty acid” means a saturated or unsaturated, straightchain or branched chain fatty acid, having from one to eighteen carbonatoms.

[0042] The term “C1 to C18 fatty acid” refers to a saturated, straightchain or branched chain fatty acid having from one to eighteen carbonatoms.

[0043] The term “divalent metal cation” refers to the ion or ions thatparticipate to form a complex with a multiplicity of protein molecules.The transition metals, the alkaline metals, and the alkaline earthmetals are examples of metals that are known to form complexes withinsulin. The transitional metals are preferred. Zinc is particularlypreferred. Other transition metals that may be pharmaceuticallyacceptable for complexing with insulin proteins include copper, cobalt,and iron.

[0044] The term “complex” has two meanings in the present invention. Inthe first, the term refers to a complex formed between one or more atomsin the proteins that form the complex and one or more divalent metalcations. The atoms in the proteins serve as electron-donating ligands.The proteins typically form a hexamer complex with divalent transitionmetal cations. The second meaning of “complex” in the present inventionis the association between the complexing compound and hexamers. The“complexing compound” is an organic molecule that typically has amultiplicity of positive charges that binds to, or complexes withhexamers in the insoluble composition, thereby stabilizing them againstdissolution. Examples of complexing compounds suitable in the presentinvention include protamine, surfen, various globin proteins [Brange,J., Galenics of Insulin, Springer-Verlag, Berlin Heidelberg (1987)], andvarious polycationic polymer compounds known to complex with insulin.

[0045] The term “protamine” refers to a mixture of strongly basicproteins obtained from fish sperm. The average molecular weight of theproteins in protamine is about 4,200 [Hoffmann, J. A., et al., ProteinExpression and Purification, 1:127-133 (1990)]. “Protamine” can refer toa relatively salt-free preparation of the proteins, often called“protamine base.” Protamine also refers to preparations comprised ofsalts of the proteins. Commercial preparations vary widely in their saltcontent.

[0046] Protamines are well-known to those skilled in the insulin art andare currently incorporated into NPH insulin products. A pure fraction ofprotamine is operable in the present invention, as well as mixtures ofproteins. Commercial preparations of protamine, however, are typicallynot homogeneous with respect to the proteins present. These arenevertheless operative in the present invention. Protamine comprised ofprotamine base is operative in the present invention, as are protaminepreparations comprised of salts of protamine, and those that aremixtures of protamine base and protamine salts. Protamine sulfate is afrequently used protamine salt.

[0047] The term “suspension” refers to a mixture of a liquid phase and asolid phase that consists of insoluble or sparingly soluble particlesthat are larger than colloidal size. Mixtures of NPH microcrystals andan aqueous solvent form suspensions. Mixtures of amorphous precipitateand an aqueous solvent also forms a suspension. The term “suspensionformulation” means a pharmaceutical composition wherein an active agentis present in a solid phase, for example, a microcrystalline solid, anamorphous precipitate, or both, which is finely dispersed in an aqueoussolvent. The finely dispersed solid is such that it may be suspended ina fairly uniform manner throughout the aqueous solvent by the action ofgently agitating the mixture, thus providing a reasonably uniformsuspension from which a dosage volume may be extracted. Examples ofcommercially available insulin suspension formulations include, forexample, NPH, PZI, and ultralente. A small proportion of the solidmatter in a microcrystalline suspension formulation may be amorphous.Preferably, the proportion of amorphous material is less than 10%, andmost preferably, less than 1% of the solid matter in a microcrystallinesuspension. Likewise, a small proportion of the solid matter in anamorphous precipitate suspension may be microcrystalline.

[0048] “NPH insulin” refers to the “Neutral Protamine Hagedorn”preparation of insulin. The meaning of such a term, and the methods forpreparing such a preparation of insulin will be familiar to the personof ordinary skill in the insulin formulation art.

[0049] The term “aqueous solvent” refers to a liquid solvent thatcontains water. An aqueous solvent system may be comprised solely ofwater, may be comprised of water plus one or more miscible solvents, andmay contain solutes. The more commonly-used miscible solvents are theshort-chain organic alcohols, such as, methanol, ethanol, propanol,short-chain ketones, such as acetone, and polyalcohols, such asglycerol.

[0050] An “isotonicity agent” is a compound that is physiologicallytolerated and imparts a suitable tonicity to a formulation to preventthe net flow of water across cell membranes that are in contact with anadministered formulation. Glycerol, which is also known as glycerin, iscommonly used as an isotonicity agent. Other isotonicity agents includesalts, e.g., sodium chloride, and monosaccharides, e.g., dextrose andlactose.

[0051] The insoluble compositions of the present invention contain ahexamer-stabilizing compound. The term “hexamer-stabilizing compound”refers to a non-proteinaceous, small molecular weight compound thatstabilizes the derivatized protein in a hexameric aggregation state.Phenolic compounds, particularly phenolic preservatives, are the bestknown stabilizing compounds for insulin and insulin derivatives. Such ahexamer-stabilizing compound stabilizes the insulin hexamer by bindingto it through specific inter-molecular contacts. Examples of suchhexamer-stabilizing agents include: various phenolic compounds, phenolicpreservatives, resorcinol, 4′-hydroxyacetanilide (tylenol),4-hydroxybenzamide, and 2,7-dihyroxynaphthalene. Multi-use formulationsof the insoluble compositions of the present invention will contain apreservative, in addition to a hexamer-stabilizing compound. Thepreservative used in formulations of the present invention may be aphenolic preservative.

[0052] The term “preservative” refers to a compound added to apharmaceutical formulation to act as an anti-microbial agent. Aparenteral formulation must meet guidelines for preservativeeffectiveness to be a commercially viable multi-use product. Amongpreservatives known in the art as being effective and acceptable inparenteral formulations are benzalkonium chloride, benzethonium,chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben,chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate,thimerosal, benzoic acid, and various mixtures thereof. See, e.g.,Wallhäusser, K.-H., Develop. Biol. Standard, 24:9-28 (1974) (S. Krager,Basel).

[0053] The term “phenolic preservative” includes the compounds phenol,m-cresol, o-cresol, p-cresol, chlorocresol, methylparaben, and mixturesthereof. Certain phenolic preservatives, such as phenol and m-cresol,are known to bind to insulin-like molecules and thereby to induceconformational changes that increase either physical or chemicalstability, or both [Birnbaum, D. T., et al., Pharmaceutical. Res.14:25-36 (1997); Rahuel-Clermont, S., et al., Biochemistry 36:5837-5845(1997)].

[0054] The term “buffer” or “pharmaceutically acceptable buffer” refersto a compound that is known to be safe for use in insulin formulationsand that has the effect of controlling the pH of the formulation at thepH desired for the formulation. The pH of the formulations of thepresent invention is from about 6.0 to about 8.0. Preferably theformulations of the present invention have a pH between about 6.8 andabout 7.8. Pharmaceutically acceptable buffers for controlling pH at amoderately acidic pH to a moderately basic pH include such compounds asphosphate, acetate, citrate, arginine, TRIS, and histidine. “TRIS”refers to 2-amino-2-hydroxymethyl-1,3,-propanediol, and to anypharmacologically acceptable salt thereof. The free base and thehydrochloride form are two common forms of TRIS. TRIS is also known inthe art as trimethylol aminomethane, tromethamine, andtris(hydroxymethyl)aminomethane. Other buffers that are pharmaceuticallyacceptable, and that are suitable for controlling pH at the desiredlevel are known to the chemist of ordinary skill.

[0055] The term “administer” means to introduce a formulation of thepresent invention into the body of a patient in need thereof to treat adisease or condition.

[0056] The term “treating” refers to the management and care of apatient having diabetes or hyperglycemia, or other condition for whichinsulin administration is indicated for the purpose of combating oralleviating symptoms and complications of those conditions. Treatingincludes administering a formulation of present invention to prevent theonset of the symptoms or complications, alleviating the symptoms orcomplications, or eliminating the disease, condition, or disorder.

[0057] As mentioned above, the present invention provides insolublecompositions that have properties similar to NPH insulin in certainrespects, and superior to NPH insulin in other respects. They aresimilar to NPH insulin in respect to their physical properties. A lightmicroscope equipped with an oil immersion objective and a crossedpolarizer was utilized to examine microcrystals comprised ofB29-Nε-octanoyl-human insulin, zinc, protamine, and phenol, preparedaccording to the method of Preparation 18. Examination at 1000×magnification showed that the B29-Nε-octanoyl-human insulinmicrocrystals were single and rod-like, exhibiting a uniform crystalmorphology. The sizes of these microcrystals fell generally within therange of approximately 2 microns long to 8 microns long. A directcomparison using this microscope showed that the morphology of thesemicrocrystals appeared to be similar to that of commerciallymanufactured pork NPH microcrystals, which has elsewhere been describedas rod-like. The size range of these B29-Nε-octanoyl-human insulinmicrocrystals was also similar to that of commercially manufactured NPHmicrocrystals, which generally have an average length of about 5microns. The commercial manufacturing specification for the mean lengthof NPH microcrystals is from 1 micron to 40 microns.

[0058] The microcrystals of the present invention are, however,unexpectedly and unpredictably different from NPH insulin crystals intheir dissolution properties, and in their time action. In particular,the microcrystals of the present invention dissolve much more slowlyunder conditions that simulate physiologic conditions than do NPHinsulin crystals, and provide a longer and flatter profile of bloodglucose control than does NPH insulin. This was demonstrated by thefollowing experiments.

[0059] Certain derivatized proteins, in soluble form, were found to havetime actions not significantly different from regular human insulin.Three groups of animals were used. Each animal in the first groupreceived a dose (0.75 nmol/kg) of Humulin® R (soluble human insulin),each animal in the second group received a dose (0.75 nmol/kg) ofsoluble B29-Nε-octanoyl-human insulin (“C8-hI”), and each animal in thethird group received a dose (0.75 nmol/kg) of solubleB29-Nε-decanoyl-human insulin (“C10-hI”). The experiments were carriedout essentially as described in Example 5, with five dogs per group. Theproteins were administered subcutaneously. Blood glucose concentrationswere determined, and are presented in the table below. TABLE 1 Bloodglucose concentrations before and after administration of Humulin ® R,soluble B29-Nε-octanoyl-human insulin (“C8-hI”), or solubleB29-Nε-decanoyl-human insulin (“C10-hI”) in normal dogs simultaneouslyadministered somatostatin to create a transient diabetic state. Valuesare mean ± standard error. Time Blood Glucose Concentration (mg/dL) (h)Humulin ® R Soluble C8-hI Soluble C10-hI −0.5 110 ± 2  115 ± 4  108 ± 2 0 101 ± 2  101 ± 7  96 ± 4  0.5 83 ± 5  80 ± 5  85 ± 6  1 54 ± 6  52 ±4  70 ± 5  1.5 49 ± 4  51 ± 2  57 ± 4  2 48 ± 4  51 ± 2  52 ± 3  2.5 55± 4  60 ± 3  56 ± 4  3 59 ± 2  65 ± 4  58 ± 4  3.5 65 ± 2  73 ± 5  63 ±4  4 71 ± 2  85 ± 6  68 ± 4  5 87 ± 2  110 ± 8  79 ± 3  6 104 ± 3  124 ±4  91 ± 7  7 119 ± 8  145 ± 14  106 ± 8  8 144 ± 5  153 ± 16  119 ± 11 

[0060] These data clearly show that soluble B29-Nε-octanoyl-humaninsulin and B29-Nε-decanoyl-human insulin, administered subcutaneouslyto normal dogs in a transient diabetic state, provide glucose loweringroughly comparable to that obtained with soluble human insulin. Mostnotably, soluble B29-Nε-octanoyl-human insulin shows a quicker onset,and shorter time action than does human insulin.

[0061] In a second experiment, the dissolution rate of crystals ofB29-Nε-octanoyl-human insulin prepared in accordance with the presentinvention was found to be markedly longer than that of a commerciallymanufactured NPH-pork insulin. This was most unexpected in view of thedata above. The dissolution rate of the NPH-pork insulin was measured byplacing 5 microliters of U100 NPH-pork insulin into 3 mL of Dulbecco'sphosphate buffered saline (without calcium or magnesium) in a 1 cm pathlength square quartz cuvette at a temperature of 22° C. This solutionwas stirred at a constant rate using a magnetic cuvette stirrer.Absorbance measurements at 320 nm were taken at 1 minute intervals. Theabsorbance at 320 nm corresponds to the light scattered by the insolubleparticles present in the aqueous suspension. Consequently, as themicrocrystals dissolve, the absorbance approaches zero. The datagenerated from this experiment are presented in FIG. 1 as the dashedline, and show that the pork NPH microcrystals were completely dissolvedafter about 1 hour.

[0062] An analogous procedure was followed to measure the dissolutionrate of B29-Nε-octanoyl-human insulin microcrystals. A volume of 12microliters of a suspension of B29-Nε-octanoyl-human insulinmicrocrystals (containing no more than 50 U/mL), prepared according tothe procedure of Preparation 18, was placed into 3 mL of Dulbecco'sphosphate buffered saline (without calcium or magnesium) in a 1 cm pathlength square quartz cuvette. This solution was stirred at the sameconstant rate and at the same temperature of 22° C. The data generatedfrom this experiment are presented in FIG. 1 as the solid line, and showthat the B29-Nε-octanoyl-human insulin microcrystals required much morethan 5 hours to dissolve.

[0063] These experiments establish that, in Dulbecco's phosphatebuffered saline (without calcium and magnesium), a solution that mimicsthe interstitial fluid in certain aspects, the rate of dissolution ofthe B29-Nε-octanoyl-human insulin microcrystals is significantly slowerthan that of pork NPH microcrystals. Again, this finding was verysurprising in light of the previous finding that solubleB29-Nε-octanoyl-human insulin had a time action actually slightlyshorter than did human insulin!

[0064] Subcutaneous interstitial fluid contains 0.3 mM human serumalbumin. Therefore, another experiment was designed to compare thedissolution rates of approximately equal quantities ofB29-Nε-octanoyl-human insulin microcrystals and pork NPH microcrystalsin Dulbecco's phosphate buffered saline containing 0.3 mM human serumalbumin.

[0065] This experiment was performed by placing 25 microliters of NPHpork insulin (approximately 3.5 mg insulin/mL) into 2 mL of Dulbecco'sphosphate buffered saline (without calcium and magnesium) containing 0.3mM human serum albumin. The resulting suspension was swirled gently byhand whereupon the microcrystals were observed to be dissolved afterabout 3 to 5 minutes.

[0066] The rate of dissolution of B29-Nε-octanoyl-human insulinmicrocrystals was observed by placing 50 microliters of aB29-Nε-octanoyl-human insulin microcrystalline formulation(approximately 1.8 mg/mL), prepared essentially as described inPreparation 18 herein, into 2 mL of Dulbecco's phosphate buffered saline(without calcium and magnesium) containing 0.3 mM human serum albumin.The resulting suspension was swirled gently by hand for about 3 to 5minutes whereupon minimal dissolution of the suspended microcrystals wasobserved to have taken place. Continued gentle stirring of this solutionusing a magnetic stirrer resulted in complete dissolution of thesuspended B29-Nε-octanoyl-human insulin microcrystals after about 2hours.

[0067] These experiments establish that the rate of dissolution of theB29-Nε-octanoyl-human insulin microcrystals is significantly slower thanthe rate of dissolution of commercially manufactured pork NPHmicrocrystals in Dulbecco's phosphate buffered saline (without calciumand magnesium) containing 0.3 mM human serum albumin.

[0068] Because the time action profile of NPH insulin preparations isrelated strongly to the rate of dissolution of the microcrystals in thesubcutaneous interstitial fluid, it is concluded from these experimentsthat the B29-Nε-octanoyl-human insulin microcrystalline suspensionformulation will possess a more protracted duration of action whenadministered subcutaneously to diabetic patients than existingcommercial NPH insulin preparations.

[0069] The insoluble compositions of the present invention may becrystals with rod-like morphology or with an irregular morphology, orthey may be amorphous precipitates. Preferred insoluble compositions arecomprised of acylated insulin or acylated insulin analog, zinc ions,which are present at about 0.3 to about 0.7 mole per mole of derivatizedprotein, a phenolic preservative selected from the group consisting ofphenol, m-cresol, o-cresol, p-cresol, chlorocresol, methylparaben, andmixtures thereof and is present in sufficient proportions with respectto the derivatized protein to facilitate formation of the R6 hexamerconformation, and protamine, which is present at about 0.15 to about 0.7mole per mole of derivatized protein.

[0070] The preferred derivatized proteins are acylated proteins, and thepreferred acylated proteins for the microcrystals and formulations ofthe present invention are fatty acid-acylated insulin, and fattyacid-acylated insulin analogs. Fatty acid-acylated human insulin ishighly preferred. Fatty acid-acylated insulin analogs are equally highlypreferred.

[0071] A preferred group of insulin analogs for preparing acylatedinsulin analogs used to form the microcrystals of the present inventionconsists of insulin analogs wherein the amino acid residue at positionB28 is Asp, Lys, Leu, Val, or Ala, the amino acid residue at positionB29 is Lys or Pro, the amino acid residue at position B10 is His or Asp,the amino acid residue at position B1 is Phe, Asp or deleted alone or incombination with a deletion of the residue at position B2, the aminoacid residue at position B30 is Thr, Ala, Ser, or deleted, and the aminoacid residue at position B9 is Ser or Asp; provided that either positionB28 or B29 is Lys.

[0072] Another preferred group of insulin analogs for use in the presentinvention consists of those wherein the isoelectric point of the insulinanalog is between about 7.0 and about 8.0. These analogs are referred toas “pI-shifted insulin analogs.” Examples of pI-shifted insulin analogsinclude, for example, ArgB31,ArgB32-human insulin,GlyA21,ArgB31,ArgB32-human insulin, ArgA0,ArgB31,ArgB32-human insulin,and ArgA0,GlyA21,ArgB31,ArgB32-human insulin.

[0073] Another preferred group of insulin analogs consists ofLysB28,ProB29-human insulin (B28 is Lys; B29 is Pro); AspB28-humaninsulin (B28 is Asp), AspB1-human insulin, ArgB31,ArgB32-human insulin,ArgA0-human insulin, AspB1,GluB13-human insulin, AlaB26-human insulin,GlyA21-human insulin, des(ThrB30)-human insulin, andGlyA21,ArgB31,ArgB32-human insulin.

[0074] Especially preferred insulin analogs include LysB28,ProB29-humaninsulin, des(ThrB30)-human insulin, AspB28-human insulin, andAlaB26-human insulin. Another especially preferred insulin analog isGlyA21, ArgB31, ArgB32-human insulin [Dörschug, M., U.S. Pat. No.5,656,722, Aug. 12, 1997]. The most preferred insulin analog isLysB28,ProB29-human insulin.

[0075] One preferred group of acylating moieties consists of fatty acidsthat are straight chain and saturated. This group consists of methanoicacid (C1), ethanoic acid (C2), propanoic acid (C3), n-butanoic acid(C4), n-pentanoic acid (C5), n-hexanoic acid (C6), n-heptanoic acid(C7), n-octanoic acid (C8), n-nonanoic acid (C9), n-decanoic acid (C10),n-undecanoic acid (C11), n-dodecanoic acid (C12), n-tridecanoic acid(C13), n-tetradecanoic acid (C14), n-pentadecanoic acid (C15),n-hexadecanoic acid (C16), n-heptadecanoic acid (C17), andn-octadecanoic acid (C18). Adjectival forms are formyl (C1), acetyl(C2), propionyl (C3), butyryl (C4), pentanoyl (C5), hexanoyl (C6),heptanoyl (C7), octanoyl (C8), nonanoyl (C9), decanoyl (C10), undecanoyl(C11), dodecanoyl (C12), tridecanoyl (C13), tetradecanoyl (C14) ormyristoyl, pentadecanoyl (C15), hexadecanoyl (C16) or palmitic,heptadecanoyl (C17), and octadecanoyl (C18).

[0076] A preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having an even number of carbonatoms—that is, C2, C4, C6, C8, C10, C12, C14, C16, and C18 saturatedfatty acids.

[0077] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having an odd number of carbonatoms—that is, C1, C3, C5, C7, C9, C11, C13, C15, and C17 saturatedfatty acids.

[0078] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having more than 5 carbon atoms—thatis, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, and C18saturated fatty acids.

[0079] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having less than 9 carbon atoms—thatis, C1, C2, C3, C4, C5, C6, C7, and C8 saturated fatty acids.

[0080] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having between 6 and 8 carbonatoms—that is, C6, C7, and C8, saturated fatty acids.

[0081] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having more than between 4 and 6carbon atoms—that is, C4, C5, and C6, saturated fatty acids.

[0082] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having more than between 2 and 4carbon atoms—that is, C2, C3, and C4, saturated fatty acids.

[0083] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having less than 6 carbon atoms—thatis, C1, C2, C3, C4, and C5 saturated fatty acids.

[0084] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having less than 4 carbon atoms—thatis, C1, C2, and C3 saturated fatty acids.

[0085] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having more than 9 carbon atoms—thatis, C10, C11, C12, C13, C14, C15, C16, C17, and C18 saturated fattyacids.

[0086] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having an even number of carbon atomsand more than 9 carbon atoms—that is, C10, C12, C14, C16, and C18saturated fatty acids.

[0087] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having 12, 14, or 16 carbon atoms,that is, C12, C14, and C16 saturated fatty acids.

[0088] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having 14 or 16 carbon atoms, that is,C14 and C16 saturated fatty acids. Fatty acids with 14 carbons areparticularly preferred. Fatty acids with 16 carbons are alsoparticularly preferred.

[0089] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of saturated fatty acids having between 4 and 10carbon atoms, that is C4, C5, C6, C7, C8, C9, and C10 saturated fattyacids.

[0090] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of saturated fatty acids having an even number ofcarbon atoms between 4 and 10 carbon atoms, that is C4, C6, C8, and C10saturated fatty acids.

[0091] Another preferred group of fatty acids for forming the fattyacid-acylated proteins used in the microcrystals of the presentinvention consists of fatty acids having between 6, 8, or 10 carbonatoms. Fatty acids with 6 carbons are particularly preferred. Fattyacids with 8 carbons are also particularly preferred. Fatty acids with10 carbons are particularly preferred.

[0092] The skilled person will appreciate that narrower preferred groupsare made by combining the preferred groups of fatty acids describedabove.

[0093] Another preferred group of acylating moieties consists ofsaturated fatty acids that are branched. A branched fatty acid has atleast two branches. The length of a “branch” of a branched fatty acidmay be described by the number of carbon atoms in the branch, beginningwith the acid carbon. For example, the branched fatty acid3-ethyl-5-methylhexanoic acid has three branches that are five, six, andsix carbons in length. In this case, the “longest” branch is sixcarbons. As another example, 2,3,4,5-tetraethyloctanoic acid has fivebranches that are 4, 5, 6, 7, and 8 carbons long. The “longest” branchis eight carbons. A preferred group of branched fatty acids are thosehaving from three to ten carbon atoms in the longest branch.

[0094] A representative number of such branched, saturated fatty acidswill be mentioned to assure the reader's comprehension of the range ofsuch fatty acids that may be used as acylating moieties of the proteinsin the present invention: 2-methyl-propioinic acid, 2-methyl-butyricacid, 3-methyl-butyric acid, 2,2-dimethyl-propionic acid,2-methyl-pentanoic acid, 3-methyl-pentanoic acid, 4-methyl-pentanoicacid, 2,2-dimethyl-butyric acid, 2,3-dimethyl-butyric acid,3,3-dimethyl-butyric acid, 2-ethyl-butyric acid, 2-methyl-hexanoic acid,5-methyl-hexanoic acid, 2,2-dimethyl-pentanoic acid,2,4-dimethyl-pentanoic acid, 2-ethyl-3-methyl-butyric acid,2-ethyl-pentanoic acid, 3-ethyl-pentanoic acid,2,2-dimethyl-3-methyl-butyric acid,2-methyl-heptanoic acid,3-methyl-heptanoic acid, 4-methyl-heptanoic acid, 5-methyl-heptanoicacid, 6-methyl-heptanoic acid, 2,2-dimethyl-hexanoic acid,2,3-dimethyl-hexanoic acid, 2,4-dimethyl-hexanoic acid,2,5-dimethyl-hexanoic acid, 3,3,-dimethyl-hexanoic acid,3,4-dimethyl-hexanoic acid, 3,5-dimethyl-hexanoic acid,4,4-dimethyl-hexanoic acid, 2-ethyl-hexanoic acid, 3- ethyl-hexanoicacid, 4-ethyl-hexanoic acid, 2-propyl-pentanoic acid, 2-ethyl-hexanoicacid, 3-ethyl-hexanoic acid, 4-ethyl-hexanoic acid,2-(1-propyl)pentanoic acid, 2-(2-propyl)pentanoic acid,2,2-diethyl-butyric acid, 2,3,4-trimethyl-pentanoic acid,2-methyl-octanoic acid, 4-methyl-octanoic acid, 7-methyl-octanoic acid,2,2-dimethyl-heptanoic acid, 2,6-dimethyl-heptanoic acid,2-ethyl-2-methyl-hexanoic acid, 3-ethyl-5-methyl-hexanoic acid,3-(1-propyl)-hexanoic acid, 2-(2-butyl)-pentanoic acid,2-(2-(2-methylpropyl))pentanoic acid, 2-methyl-nonanoic acid,8-methyl-nonanoic acid, 6-ethyl-octanoic acid, 4-(1-propyl)-heptanoicacid, 5-(2-propyl)-heptanoic acid, 3-methyl-undecanoic acid,2-pentyl-heptanoic acid, 2,3,4,5,6-pentamethyl-heptanoic acid,2,6-diethyl-octanoic acid, 2-hexyl-octanoic acid,2,3,4,5,6,7-hexamethyl-octanoic acid, 3,3-diethyl-4,4-diethyl-hexanoicacid, 2-heptyl-nonanoic acid, 2,3,4,5-tetraethyl-octanoic acid,2-octyl-decanoic acid, and2-(1-propyl)-3-(1-propyl)-4,5-diethyl-6-methyl-heptanoic acid.

[0095] Yet another preferred group of acylating moieties consists ofcyclic alkyl acids having from 5 to 24 carbon atoms, wherein the cyclicalkyl moiety, or moieties, have 5 to 7 carbon atoms. A representativenumber of such cyclic alkyl acids will be mentioned to assure thereader's comprehension of the range of such acids that may be used asacylating moieties of the proteins in the present invention:cyclopentyl-formic acid, cyclohexyl-formic acid, 1-cyclopentyl-aceticacid, 2-cyclohexyl-acetic acid, 1,2-dicyclopentyl-acetic acid, and thelike.

[0096] A preferred group of derivatized proteins for use in themicrocrystals of the present invention consists of mono-acylatedproteins. Mono-acylation at the ε-amino group is most preferred. Forinsulin, mono-acylation at LysB29 is preferred. Similarly, for certaininsulin analogs, such as, LysB28,ProB29-human insulin analog,mono-acylation at the ε-amino group of LysB28 is most preferred.Mono-acylation at the α-amino group of the B-chain (B1) is alsopreferred. Mono-acylation at the α-amino group of the A-chain (A1) isalso preferred.

[0097] Another preferred group of acylated proteins for use in themicrocrystals of the present invention consists of di-acylated proteins.The di-acylation may be, for example, at the ε-amino group of Lys and atthe α-amino group of the B-chain, or may be at the ε-amino group of Lysand at the α-amino group of the A-chain, or may be at the α-amino groupthe A-chain and at the α-amino group of the B-chain.

[0098] Another preferred group of acylated proteins for use in themicrocrystals of the present invention consists of tri-acylatedproteins. Tri-acylated proteins are those that are acylated at theε-amino group of Lys, at the α-amino group of the B-chain, and at theα-amino group of the A-chain.

[0099] It is also preferred to use acylated proteins that are a mixtureof mono-acylated and di-acylated proteins.

[0100] It is likewise preferred to use acylated proteins that are amixture of mono-acylated and tri-acylated proteins.

[0101] Another preferred group of acylated proteins consists of amixture of di-acylated and tri-acylated proteins.

[0102] Also preferred is to use acylated proteins that are a mixture ofmono-acylated, di-acylated, and tri-acylated proteins.

[0103] Certain fatty acid-acylated proteins used in the presentmicrocrystals will be mentioned to assure the reader's comprehension ofthe scope of the present invention. The list is illustrative, and thefact that a particular fatty acid-acylated protein is not mentioned doesnot mean that a microcrystal containing it is not within the scope ofthe present invention.

[0104] B29-Nε-Formyl-human insulin.

[0105] B1-Nα-Formyl-human insulin.

[0106] A1-Nα-Formyl-human insulin.

[0107] B29-Nε-Formyl-,B1-Nα-formyl-human insulin.

[0108] B29-Nε-Formyl-, A1-Nα-formyl-human insulin.

[0109] A1-Nα-Formyl-,B1-Nα-formyl-human insulin.

[0110] B29-Nε-Formyl-, A1-Nα-formyl-, B1-Nα-formyl-human insulin.

[0111] B29-Nε-Acetyl-human insulin.

[0112] B1-Nα-Acetyl-human insulin.

[0113] A1-Nα-Acetyl-human insulin.

[0114] B29-Nε-Acetyl-, B1-Nα-acetyl-human insulin.

[0115] B29-Nε-Acetyl-, A1-Nα-acetyl-human insulin.

[0116] A1-Nα-Acetyl-, B1-Nα-acetyl-human insulin.

[0117] B29-Nε-Acetyl-, A1-Nα-acetyl-, B1-Nα-acetyl-human insulin.

[0118] B29-Nε-Propionyl-human insulin.

[0119] B1-Nα-Propionyl-human insulin.

[0120] A1-Nα-Propionyl-human insulin.

[0121] B29-Nε-Propionyl-, B1-Nα-propionyl-human insulin.

[0122] B29-Nε-Propionyl-, A1-Nα-propionyl-human insulin.

[0123] A1-Nα-Propionyl-, B1-Nα-propionyl-human insulin.

[0124] B29-Nε-Propionyl-, A1-Nα-propionyl-, B1-Nα-propionyl-humaninsulin.

[0125] B29-Nε-Butyryl-human insulin.

[0126] B1-Nα-Butyryl-human insulin.

[0127] A1-Nα-Butyryl-human insulin.

[0128] B29-Nε-Butyryl-,B1-Nα-butyryl-human insulin.

[0129] B29-Nε-Butyryl-,A1-Nα-butyryl-human insulin.

[0130] A1-Nα-Butyryl-,B1-Nα-butyryl-human insulin.

[0131] B29-Nε-Butyryl-, A1-Nα-butyryl-,B1-Nα-butyryl-human insulin.

[0132] B29-Nε-Pentanoyl-human insulin.

[0133] B1-Nα-Pentanoyl-human insulin.

[0134] A1-Nα-Pentanoyl-human insulin.

[0135] B29-Nε-Pentanoyl-,B1-Nα-pentanoyl-human insulin.

[0136] B29-Nε-Pentanoyl-,A1-Nα-pentanoyl-human insulin.

[0137] A1-Nα-Pentanoyl-,B1-Nα-pentanoyl-human insulin.

[0138] B29-Nε-Pentanoyl-, A1-Nα-pentanoyl-,B1-Nα-pentanoyl-humaninsulin.

[0139] B29-Nε-Hexanoyl-human insulin.

[0140] B1-Nα-Hexanoyl-human insulin.

[0141] A1-Nα-Hexanoyl-human insulin.

[0142] B29-Nε-Hexanoyl-,B1-Nα-hexanoyl-human insulin.

[0143] B29-Nε-Hexanoyl-,A1-Nα-hexanoyl-human insulin.

[0144] A1-Nα-Hexanoyl-,B1-Nα-hexanoyl-human insulin.

[0145] B29-Nε-Hexanoyl-, A1-Nα-hexanoyl-,B1-Nα-hexanoyl-human insulin.

[0146] B29-Nε-Heptanoyl-human insulin.

[0147] B1-Nα-Heptanoyl-human insulin.

[0148] A1-Nα-Heptanoyl-human insulin.

[0149] B29-Nε-Heptanoyl-,B1-Nα-heptanoyl-human insulin.

[0150] B29-Nε-Heptanoyl-,A1-Nα-heptanoyl-human insulin.

[0151] A1-Nα-Heptanoyl-,B1-Nα-heptanoyl-human insulin.

[0152] B29-Nε-Heptanoyl-, A1-Nα-heptanoyl-,B1-Nα-heptanoyl-humaninsulin.

[0153] B29-Nε-Octanoyl-human insulin.

[0154] B1-Nα-Octanoyl-human insulin.

[0155] A1-Nα-Octanoyl-human insulin.

[0156] B29-Nε-Octanoyl-,B1-Nα-octanoyl-human insulin.

[0157] B29-Nε-Octanoyl-,A1-Nα-octanoyl-human insulin.

[0158] A1-Nα-Octanoyl-,B1-Nα-octanoyl-human insulin.

[0159] B29-Nε-Octanoyl-, A1-Nα-octanoyl-,B1-Nα-octanoyl-human insulin.

[0160] B29-Nε-Nonanoyl-human insulin.

[0161] B1-Nα-Nonanoyl-human insulin.

[0162] A1-Nα-Nonanoyl-human insulin.

[0163] B29-Nε-Nonanoyl-,B1-Nα-nonanoyl-human insulin.

[0164] B29-Nε-Nonanoyl-,A1-Nα-nonanoyl-human insulin.

[0165] A1-Nα-Nonanoyl-,B1-Nα-nonanoyl-human insulin.

[0166] B29-Nε-Nonanoyl-, A1-Nα-nonanoyl-,B1-Nα-nonanoyl-human insulin.

[0167] B29-Nε-Decanoyl-human insulin.

[0168] B1-Nα-Decanoyl-human insulin.

[0169] A1-Nα-Decanoyl-human insulin.

[0170] B29-Nε-Decanoyl-,B1-Nα-decanoyl-human insulin.

[0171] B29-Nε-Decanoyl-,A1-Nα-decanoyl-human insulin.

[0172] A1-Nα-Decanoyl-,B1-Nα-decanoyl-human insulin.

[0173] B29-Nε-Decanoyl-,A1-Nα-decanoyl-, B1-Nα-decanoyl-human insulin.

[0174] B28-Nε-Formyl-LysB28,ProB29-human insulin analog.

[0175] B1-Nα-Formyl-LysB28,ProB29-human insulin analog.

[0176] A1-Nα-Formyl-LysB28,ProB29-human insulin analog.

[0177] B28-Nε-Formyl-, B1-Nα-formyl-LysB28,ProB29-human insulin analog.

[0178] B28-Nε-Formyl-, A1-Nα-formyl-LysB28,ProB29-human insulin analog.

[0179] A1-Nα-Formyl-,B1-Nα-formyl-LysB28,ProB29-human insulin analog.

[0180] B28-Nε-Formyl-, A1-Nα-formyl-, B1-Nα-formyl-LysB28,ProB29-humaninsulin analog.

[0181] B28-Nε-Acetyl-LysB28,ProB29-human insulin analog.

[0182] B1-Nα-Acetyl-LysB28,ProB29-human insulin analog.

[0183] A1-Nα-Acetyl-LysB28,ProB29-human insulin analog.

[0184] B28-Nε-Acetyl-, B1-Nα-acetyl-LysB28,ProB29-human insulin analog.

[0185] B28-Nε-Acetyl-, A1-Nα-acetyl-LysB28,ProB29-human insulin analog.

[0186] A1-Nα-Acetyl-, B1-Nα-acetyl-LysB28,ProB29-human insulin analog.

[0187] B28-Nε-Acetyl-, A1-Nα-acetyl-, B1-Nα-acetyl-LysB28,ProB29-humaninsulin analog.

[0188] B28-Nε-Propionyl-LysB28,ProB29-human insulin analog.

[0189] B1-Nα-Propionyl-LysB28,ProB29-human insulin analog.

[0190] A1-Nα-Propionyl-LysB28,ProB29-human insulin analog.

[0191] B28-Nε-Propionyl-, B1-Nα-propionyl-LysB28,ProB29-human insulinanalog.

[0192] B28-Nε-Propionyl-, A1-Nα-propionyl-LysB28,ProB29-human insulinanalog.

[0193] A1-Nα-Propionyl-,B1-Nα-propionyl-LysB28,ProB29-human insulinanalog.

[0194] B28-Nε-Propionyl-, A1-Nα-propionyl-,B1-Nα-propionyl-LysB28,ProB29-human insulin analog.

[0195] B28-Nε-Butyryl-LysB28,ProB29-human insulin analog.

[0196] B1-Nα-Butyryl-LysB28,ProB29-human insulin analog.

[0197] A1-Nα-Butyryl-LysB28,ProB29-human insulin analog.

[0198] B28-Nε-Butyryl-,B1-Nα-butyryl-LysB28,ProB29-human insulin analog.

[0199] B28-Nε-Butyryl-,A1-Nα-butyryl-LysB28,ProB29-human insulin analog.

[0200] A1-Nα-Butyryl-,B1-Nα-butyryl-LysB28,ProB29-human insulin analog.

[0201] B28-Nε-Butyryl-, A1-Nα-butyryl-,B1-Nα-butyryl-LysB28,ProB29-human insulin analog.

[0202] B28-Nε-Pentanoyl-LysB28,ProB29-human insulin analog.

[0203] B1-Nα-Pentanoyl-LysB28,ProB29-human insulin analog.

[0204] A1-Nα-Pentanoyl-LysB28,ProB29-human insulin analog.

[0205] B28-Nε-Pentanoyl-, B1-Nα-pentanoyl-LysB28,ProB29-human insulinanalog.

[0206] B28-Nε-Pentanoyl-, A1-Nα-pentanoyl-LysB28,ProB29-human insulinanalog.

[0207] A1-Nα-Pentanoyl-,B1-Nα-pentanoyl-LysB28,ProB29-human insulinanalog.

[0208] B28-Nε-Pentanoyl-,A1-Nα-pentanoyl-,B1-Nα-pentanoyl-LysB28,ProB29-human insulin analog.

[0209] B28-Nε-Hexanoyl-LysB28,ProB29-human insulin analog.

[0210] B1-Nα-Hexanoyl-LysB28,ProB29-human insulin analog.

[0211] A1-Nα-Hexanoyl-LysB28,ProB29-human insulin analog.

[0212] B28-Nε-Hexanoyl-,B1-Nα-hexanoyl-LysB28,ProB29-human insulinanalog.

[0213] B28-Nε-Hexanoyl-,A1-Nα-hexanoyl-LysB28,ProB29-human insulinanalog.

[0214] A1-Nα-Hexanoyl-,B1-Nα-hexanoyl-LysB28,ProB29-human insulinanalog.

[0215] B28-Nε-Hexanoyl-,A1-Nα-hexanoyl-,B1-Nα-hexanoyl-LysB28,ProB29-human insulin analog.

[0216] B28-Nε-Heptanoyl-LysB28,ProB29-human insulin analog.

[0217] B1-Nα-Heptanoyl-LysB28,ProB29-human insulin analog.

[0218] A1-Nα-Heptanoyl-LysB28,ProB29-human insulin analog.

[0219] B28-Nε-Heptanoyl- B1-Nα-heptanoyl-LysB28,ProB29-human insulinanalog.

[0220] B28-Nε-Heptanoyl-,A1-Nα-heptanoyl-LysB28,ProB29-human insulinanalog.

[0221] A1-Nα-Heptanoyl-,B1-Nα-heptanoyl-LysB28,ProB29-human insulinanalog.

[0222] B28-Nε-Heptanoyl-,A1-Nα-heptanoyl-,B1-Nα-heptanoyl-LysB28,ProB29-human insulin analog.

[0223] B28-Nε-Octanoyl-LysB28,ProB29-human insulin analog.

[0224] B1-Nα-Octanoyl-LysB28,ProB29-human insulin analog.

[0225] A1-Nα-Octanoyl-LysB28,ProB29-human insulin analog.

[0226] B28-Nε-Octanoyl-,B1-Nα-octanoyl-LysB28,ProB29-human insulinanalog.

[0227] B28-Nε-Octanoyl-,A1-Nα-octanoyl-LysB28,ProB29-human insulinanalog.

[0228] A1-Nα-Octanoyl-,B1-Nα-octanoyl-LysB28,ProB29-human insulinanalog.

[0229] B28-Nε-Octanoyl-,A1-Nα-octanoyl-,B1-Nα-octanoyl-LysB28,ProB29-human insulin analog.

[0230] B28-Nε-Nonanoyl-LysB28,ProB29-human insulin analog.

[0231] B1-Nα-Nonanoyl-LysB28,ProB29-human insulin analog.

[0232] A1-Nα-Nonanoyl-LysB28,ProB29-human insulin analog.

[0233] B28-Nε-Nonanoyl-,B1-Nα-nonanoyl-LysB28,ProB29-human insulinanalog.

[0234] B28-Nε-Nonanoyl-,A1-Nα-nonanoyl-LysB28,ProB29-human insulinanalog.

[0235] A1-Nα-Nonanoyl-,B1-Nα-nonanoyl-LysB28,ProB29-human insulinanalog.

[0236] B28-Nε-Nonanoyl-,A1-Nα-nonanoyl-,B1-Nα-nonanoyl-LysB28,ProB29-human insulin analog.

[0237] B28-Nε-Decanoyl-LysB28,ProB29-human insulin analog.

[0238] B1-Nα-Decanoyl-LysB28,ProB29-human insulin analog.

[0239] A1-Nα-Decanoyl-LysB28,ProB29-human insulin analog.

[0240] B28-Nε-Decanoyl-,B1-Nα-decanoyl-LysB28,ProB29-human insulinanalog.

[0241] B28-Nε-Decanoyl-,A1-Nα-decanoyl-LysB28,ProB29-human insulinanalog.

[0242] A1-Nα-Decanoyl-,B1-Nα-decanoyl-LysB28,ProB29-human insulinanalog.

[0243]B28-Nε-Decanoyl-,A1-Nα-decanoyl-,B1-Nα-decanoyl-LysB28,ProB29-humaninsulin analog.

[0244] B29-Nε-Pentanoyl-GlyA21,ArgB31,ArgB32-human insulin.

[0245] B1-Nα-Hexanoyl-GlyA21,ArgB31,ArgB32-human insulin.

[0246] A1-Nα-Heptanoyl-GlyA21,ArgB31,ArgB32-human insulin.

[0247] B29-Nε-Octanoyl-, B1-Nα-octanoyl-GlyA21,ArgB31,ArgB32-humaninsulin.

[0248] B29-Nε-Propionyl-, A1-Nα-propionyl-GlyA21,ArgB31,ArgB32-humaninsulin.

[0249] A1-Nα-Acetyl, B1-Nα-acetyl-GlyA21,ArgB31,ArgB32-human insulin.

[0250] B29-Nε-Formyl-,A1-Nα-formyl-,B1-Nα-formyl-GlyA21,ArgB31,ArgB32-human insulin.

[0251] B29-Nε-Formyl-des (TyrB26) -human insulin.

[0252] B1-Nα-Acetyl-AspB28-human insulin.

[0253] B29-Nε-Propionyl-,A1-Nα-propionyl-,B1-Nα-propionyl-AspB1,AspB3,AspB21-human insulin.

[0254] B29-Nε-Pentanoyl-GlyA21-human insulin.

[0255] B1-Nα-Hexanoyl-GlyA21-human insulin.

[0256] A1-Nα-Heptanoyl-GlyA21-human insulin.

[0257] B29-Nε-Octanoyl-,B1-Nα-octanoyl-GlyA21-human insulin.

[0258] B29-Nε-Propionyl-, A1-Nα-propionyl-GlyA21-human insulin.

[0259] A1-Nα-Acetyl, B1-Nα-acetyl-GlyA21-human insulin.

[0260] B29-Nε-Formyl-, A1-Nα-formyl-,B1-Nα-formyl-GlyA21-human insulin.

[0261] B29-Nε-Butyryl-des(ThrB30)-human insulin.

[0262] B1-Nα-Butyryl-des(ThrB30)-human insulin.

[0263] A1-Nα-Butyryl-des(ThrB30)-human insulin.

[0264] B29-Nε-Butyryl-,B1-Nα-butyryl-des(ThrB30)-human insulin.

[0265] B29-Nε-Butyryl-,A1-Nα-butyryl-des(ThrB30)-human insulin.

[0266] A1-Nα-Butyryl-,B1-Nα-butyryl-des(ThrB30)-human insulin.

[0267] B29-Nε-Butyryl-, A1-Nα-butyryl-,B1-Nα-butyryl-des (ThrB30)-humaninsulin.

[0268] Aqueous compositions containing water as the major solvent arepreferred. Aqueous suspensions wherein water is the solvent are highlypreferred.

[0269] The compositions of the present invention are used to treatpatients who have diabetes or hyperglycemia. The formulations of thepresent invention will typically provide derivatized protein atconcentrations of from about 1 mg/mL to about 10 mg/mL. Presentformulations of insulin products are typically characterized in terms ofthe concentration of units of insulin activity (units/mL), such as U40,U50, U100, and so on, which correspond roughly to about 1.4, 1.75, and3.5 mg/mL preparations, respectively. The dose, route of administration,and the number of administrations per day will be determined by aphysician considering such factors as the therapeutic objectives, thenature and cause of the patient's disease, the patient's gender andweight, level of exercise, eating habits, the method of administration,and other factors known to the skilled physician. In broad range, adaily dose would be in the range of from about 1 nmol/kg body weight toabout 6 nmol/kg body weight (6 nmol is considered equivalent to about 1unit of insulin activity). A dose of between about 2 and about 3 nmol/kgis typical of present insulin therapy.

[0270] The physician of ordinary skill in treating diabetes will be ableto select the therapeutically most advantageous means to administer theformulations of the present invention. Parenteral routes ofadministration are preferred. Typical routes of parenteraladministration of suspension formulations of insulin are thesubcutaneous and intramuscular routes. The compositions and formulationsof the present invention may also be administered by nasal, buccal,pulmonary, or occular routes.

[0271] Glycerol at a concentration of 12 mg/mL to 25 mg/mL is preferredas an isotonicity agent. Yet more highly preferred for isotonicity is touse glycerol at a concentration of from about 15 mg/mL to about 17mg/mL.

[0272] M-cresol and phenol, or mixtures thereof, are preferredpreservatives in formulations of the present invention.

[0273] Insulin, insulin analogs, or proinsulins used to preparederivatized proteins can be prepared by any of a variety of recognizedpeptide synthesis techniques including classical (solution) methods,solid phase methods, semi-synthetic methods, and more recent recombinantDNA methods. For example, see Chance, R. E., et al., U.S. Pat. No.5,514,646, May 7, 1996; EPO publication number 383,472, Feb. 7, 1996;Brange, J. J. V., et al. EPO publication number 214,826, Mar. 18, 1987;and Belagaje, R. M., et al., U.S. Pat. No. 5,304,473, Apr. 19, 1994,which disclose the preparation of various proinsulin and insulinanalogs. These references are expressly incorporated herein byreference.

[0274] Generally, derivatized proteins are prepared using methods knownin the art. The publications listed above to describe derivatizedproteins contain suitable methods to prepare derivatized proteins. Thosepublications are expressly incorporated by reference for methods ofpreparing derivatized proteins. To prepare acylated proteins, theprotein is reacted with an activated organic acid, such as an activatedfatty acid. Activated fatty acids are derivatives of commonly employedacylating agents, and include activated esters of fatty acids, fattyacid halides, activated amides of fatty acids, such as, activatedazolide derivatives [Hansen, L. B., WIPO Publication No. 98/02460, Jan.22, 1998], and fatty acid anhydrides. The use of activated esters,especially N-hydroxysuccinimide esters of fatty acids, is a particularlyadvantageous means of acylating a free amino acid with a fatty acid.Lapidot, et al. describe the preparation of N-hydroxysuccinimide estersand their use in the preparation of N-lauroyl-glycine,N-lauroyl-L-serine, and N-lauroyl-L-glutamic acid. The term “activatedfatty acid ester” means a fatty acid which has been activated usinggeneral techniques known in the art [Riordan, J. F. and Vallee, B. L.,Methods in Enzymology, XXV:494-499 (1972); Lapidot, Y., et al., J. LipidRes. 8:142-145 (1967)]. Hydroxybenzotriazide (HOBT),N-hydroxysuccinimide and derivatives thereof are particularly well knownfor forming activated acids for peptide synthesis.

[0275] To selectively acylate the ε-amino group, various protectinggroups may be used to block the α-amino groups during the coupling. Theselection of a suitable protecting group is known to one skilled in theart and includes p-methoxybenzoxycarbonyl (pmZ). Preferably, the ε-aminogroup is acylated in a one-step synthesis without the use ofamino-protecting groups. A process for selective acylation at theNε-amino group of Lys is disclosed and claimed by Baker, J. C., et al.,U.S. Pat. No. 5,646,242, Jul. 8, 1997, the entire disclosure of which isincorporated expressly by reference. A process for preparing a drypowder of an acylated protein is disclosed and claimed by Baker, J. C.,et al., U.S. Pat. No. 5,700,904, Dec. 23, 1997, the entire disclosure ofwhich is incorporated herein expressly by reference.

[0276] The primary role of zinc in the present invention is tofacilitate formation of Zn(II) hexamers of the derivatized protein. Zincis known to facilitate the formation of hexamers of insulin, and ofinsulin analogs. Zinc likewise promotes the formation of hexamers ofderivatized insulin and insulin analogs. Hexamer formation isconveniently achieved by bringing the pH of a solution comprisingderivatized protein into the neutral region in the presence of Zn(II)ions, or by adding Zn(II) after the pH has been adjusted to the neutralregion.

[0277] For efficient yield of microcrystals or amorphous precipitate,the ratio of zinc to derivatized protein in the microcrystal andamorphous precipitate of the present invention is bounded at the lowerlimit by about 0.33, that is, two zinc atoms per hexamer of derivatizedprotein which are needed for efficient hexamerization. The microcrystaland amorphous precipitate compositions will form suitably with about 2to about 4-6 zinc atoms present. Even more zinc may be used during theprocess if a compound that competes with the protein for zinc binding,such as citrate or phosphate, is present. Excess zinc above the amountneeded for hexamerization may be desirable to more strongly drivehexamerization. Also, excess zinc above the amount needed forhexamerization can be present in a formulation of the present invention,and may be desirable to improve chemical and physical stability, toimprove suspendability, and possibly to extend time-action further.Consequently there is a fairly wide range of zinc:protein ratiosallowable in the formulations of the present invention.

[0278] In accordance with the present invention, zinc is present in theformulation in an amount of from about 0.3 mole to about 7 moles permole of derivatized protein and more preferably about from 0.3 mole toabout 1.0 mole of derivatized protein. Yet more highly preferred is aratio of zinc to derivatized protein from about 0.3 to about 0.7 mole ofzinc atoms per mole of derivatized protein. Most highly preferred is aratio of zinc to derivatized protein from about 0.30 to about 0.55 moleof zinc atoms per mole of derivatized protein. For higher zincformulations that are similar to PZI preparations, the zinc ratio isfrom about 5 to about 7 moles of zinc per mole of derivatized protein.

[0279] The zinc compound that provides zinc for the present inventionmay be any pharmaceutically acceptable zinc compound. The addition ofzinc to insulin preparations is known in the art, as arepharmaceutically acceptable sources of zinc. Preferred zinc compounds tosupply zinc for the present invention include zinc chloride, zincacetate, zinc citrate, zinc oxide, and zinc nitrate.

[0280] A complexing compound is required for the microcrystals andprecipitates of the present invention. The complexing compound must bepresent in sufficient quantities to cause substantial precipitation andcrystallization of hexamers of the derivatized protein. Such quantitiescan be readily determined for a particular preparation of a particularcomplexing compound by simple titration experiments. Ideally, thecomplexing compound concentration is adjusted so that there isnegligible complexing compound remaining in the soluble phase aftercompletion of precipitation and crystallization. This requires combiningthe complexing compound based on an experimentally determined “isophane”ratio. This ratio is expected to be very similar to that of NPH and NPL.However, it may be slightly different because acylation may affect thenature of the protein-protamine interaction.

[0281] When protamine is the complexing compound, it is present in themicrocrystal in an amount of from about 0.15 mg to about 0.5 mg per 3.5mg of the derivatized protein. The ratio of protamine to derivatizedprotein is preferably from about 0.25 to about 0.40 (mg/mg). Morepreferably the ratio is from about 0.25 to about 0.38 (mg/mg).Preferably, protamine is in an amount of 0.05 mg to about 0.2 mg per mgof the derivatized protein, and more preferably, from about 0.05 toabout 0.15 milligram of protamine per milligram of derivatized protein.Protamine sulfate is the preferred salt form of protamine for use in thepresent invention.

[0282] To further extend the time action of the compositions of thepresent invention or to improve their suspendability, additionalprotamine and zinc may be added after crystallization. Thus, also withinthe present invention are formulations having protamine at higher thanisophane ratios. For these formulations, the protamine ratio is from0.25 mg to about 0.5 mg of protamine per mg of derivatized protein.

[0283] A required component of the microcrystals and precipitates of thepresent invention is a hexamer stabilizing compound. The structures ofthree hexameric conformations have been characterized in the literature,and are designated T6, T3R3, and R6. In the presence of hexamerstabilizing compound, such as various phenolic compounds, the R6conformation is stabilized. Therefore, it is highly likely that hexamersare in the R6 conformation, or the T3R3 conformation in the crystals andprecipitates produced in the presence of a hexamer stabilizing compound,such as phenol. A wide range of hexamer stabilizing compounds aresuitable. At least 2 moles of hexamer stabilizing compound per hexamerof derivatized protein are required for effective hexamer stabilization.It is preferred that at least 3 moles of hexamer stabilizing compoundper hexamer of derivatized protein be present in the microcrystals andprecipitates of the present invention. The presence of higher ratios ofhexamer stabilizing compound, at least up to 25 to 50-fold higher, inthe solution from which the microcrystals and precipitates are preparedwill not adversely affect hexamer stabilization.

[0284] In formulations of the present invention, a preservative may bepresent, especially if the formulation is intended to be sampled frommultiple times. As mentioned above, a wide range of suitablepreservatives are known. Preferably, the preservative is present in thesolution in an amount suitable to provide an antimicrobial effectsufficient to meet pharmacopoeial requirements.

[0285] Preferred preservatives are the phenolic preservatives, which areenumerated above. Preferred concentrations for the phenolic preservativeare from about 2 mg to about 5 mg per milliliter of the aqueoussuspension formulation. These concentrations refer to the total mass ofphenolic preservatives because mixtures of individual phenolicpreservatives are contemplated. Suitable phenolic preservatives include,for example, phenol, m-cresol, and methylparaben. Preferred phenoliccompounds are phenol and m-cresol. Mixtures of phenolic compounds, suchas phenol and m-cresol, are also contemplated and highly preferred.Examples of mixtures of phenolic compounds are 0.6 mg/mL phenol and 1.6mg/mL m-cresol, and 0.7 mg/mL phenol and 1.8 mg/mL m-cresol.

[0286] The microcrystals of the present invention are preferablyoblong-shaped, single crystals composed of derivatized protein complexedwith a divalent cation, and including a complexing compound and ahexamer-stabilizing compound. The mean length of the microcrystals ofthe present invention preferably is within the range of 1 micron to 40microns, and more preferably is within the size range of 3 microns to 15microns.

[0287] A preferred composition comprises from about 3 mg to about 6 mgof protamine sulfate per 35 mg of derivatized protein, and from about0.1 to about 0.4 mg zinc per 35 mg of derivatized protein. Anotherpreferred composition comprises from about 10 mg to about 17 mg ofprotamine sulfate per 35 mg of derivatized protein, and from about 2.0to about 2.5 mg zinc per 35 mg of derivatized protein. Another preferredcomposition comprises, per mL, protamine sulfate, 0.34-0.38 mg; zinc,0.01-0.04 mg; and derivatized protein, 3.2-3.8 mg.

[0288] The use of the present insoluble compositions to prepare amedicament for the treatment of diabetes or hyperglycemia is alsocontemplated. The amorphous precipitates and crystals of the presentinvention can be prepared for use in medicaments, or other used, by manydifferent processes. In summary, suitable processes will generallyfollow the sequence: solubilization, hexamerization, complexation,precipitation, crystallization, and optionally formulation.Solubilization means the dissolution of derivatized protein sufficientlyto allow it to form hexamers. Hexamerization refers to the processwherein molecules of derivatized protein bind with zinc(II) atoms toform hexamers. Complexation denotes the formation of insoluble complexesbetween the hexamers and protamine. Precipitation results typically fromthe formation of insoluble complexes. Crystallization involves theconversion of precipitated hexamer/protamine complexes into crystals,typically, rod-like crystals.

[0289] Solubilization is carried out by dissolving the derivatizedprotein in an aqueous solvent. The aqueous solvent may be, for example,an acidic solution, a neutral solution, or a basic solution. The aqueoussolvent may be comprised partially of a miscible organic solvent, suchas ethanol, acetonitrile, dimethylsulfoxide, and the like. Acidicsolutions may be, for example, solutions of HCl, advantageously fromabout 0.01 N HCl to about 1.0 N HCl. Other acids that arepharmaceutically acceptable may be employed as well. Basic solutions maybe, for example, solutions of NaOH, advantageously from about 0.01 NNaOH to about 1.0 N NaOH, or higher. Other bases that arepharmaceutically acceptable may be employed as well. For the sake ofprotein stability, the concentration of acid or base is preferably aslow as possible while still being effective to adequately dissolve thederivatized protein.

[0290] Many derivatized proteins may be dissolved at neutral pH.Solutions to dissolve derivatized proteins at neutral pH may contain abuffer and optionally, salts, a phenolic compound or compounds, zinc, oran isotonicity agent.

[0291] The solution conditions required for hexamerization are thosethat allow the formation of derivatized protein-zinc hexamers insolution. Typically, hexamerization requires zinc and neutral pH. Thepresence of a hexamer-stabilizing compound advantageously influences theconformation of the derivatized protein in the hexamer, and promotes theR6 or the T3R3 hexamer conformations.

[0292] The complexation step must involve the combination of protaminewith hexamer under solution conditions where each is initially soluble.This could be accomplished by combining separate solutions of hexamericderivatized protein and of protamine, or by forming a solution ofderivatized protein and protamine at acidic or basic pH, and thenshifting the pH to the neutral range.

[0293] During crystallization, the solution conditions must stabilizethe crystallizing species. Thus, the solution conditions will determinethe rate and outcome of the crystallization. Crystallization likelyoccurs through an equilibrium involving non-crystalline precipitatedderivatized-protamine complexes, dissolved derivatized protein-protaminecomplex, and crystallized derivatized protein-protamine. The conditionschosen for crystallization drive the equilibrium toward crystalformation. Also, in light of the hypothesized equilibrium, thesolubility of the derivatized protein is expected to profoundly affectrate and size because a lower solubility will slow the net conversionfrom precipitate to solution to crystal. Furthermore, it iswell-recognized that slowing the rate of crystallization often resultsin larger crystals. Thus, the crystallization rate and crystal size arethought to depend on the size and nature of the derivatizing moiety onthe derivatized protein.

[0294] Crystallization parameters that influence the crystallizationrate and the size of crystals of the present invention are: acyl groupsize and nature; temperature; the presence and concentration ofcompounds that compete with derivatized protein for zinc, such ascitrate, phosphate, and the like; the nature and concentration ofphenolic compound(s); zinc concentration; the presence and concentrationof a miscible organic solvent; the time permitted for crystallization;the pH and ionic strength; buffer identity and concentration; theconcentration of precipitants; the presence of seeding materials; theshape and material of the container; the stirring rate; and the totalprotein concentration. Temperature and the concentration of competingcompounds are thought to be of particular importance.

[0295] Competing compounds, such as citrate, affect the rate at whichcrystals form, and indirectly, crystal size and quality. These compoundsmay exert their effect by forming coordination complexes with zinc insolution, thus competing with the relatively weak zinc binding sites onthe surface of derivatized protein hexamer for zinc. Occupation of theseweak surface binding sites probably impedes crystallization.Additionally, many derivatized proteins are partially insoluble in thepresence of little more than 0.333 zinc per mole of derivatized protein,and the presence of competing compounds restores solubility, and permitscrystallization. The exact concentration of competing compound will needto be optimized for each derivatized protein. As an upper limit, ofcourse, is the concentration at which zinc is precipitated by thecompeting compound, or the concentration at which residual competingcompound would be pharmaceutically unacceptable, such as, when it wouldcause pain or irritation at the site of administration.

[0296] An example of a process for preparing the precipitates andcrystals of the present invention follows. A measured amount of a powderof the derivatized protein is dissolved in an aqueous solvent containinga phenolic preservative. To this solution is added a solution of zinc asone of its soluble salts, for example Zn(II)Cl₂, to provide from about0.3 moles of zinc per mole of derivatized insulin to about 0.7 moles, orto as much as 1.0 moles, of zinc per mole of derivatized insulin.Absolute ethanol, or another miscible organic solvent, may optionally beadded to this solution in an amount to make the solution from about 5%to about 10% by volume organic solvent. This solution may then befiltered through a 0.22 micron, low-protein binding filter. A secondsolution is prepared by dissolving a measured amount of protamine inwater equal to one tenth the concentration by weight of theaforementioned derivatized insulin solution. This solution is filteredthrough a 0.22 micron, low-protein binding filter. The derivatizedinsulin solution and the protamine solutions are combined in equalvolumes, and the resulting solution is then stirred slowly at roomtemperature (typically about 20-25° C.) whereupon the microcrystals ofthe derivatized protein are formed within a period from about 12 hoursto about 10 days.

[0297] The microcrystals may then be separated from the mother liquorand introduced into a different solvent, for storage and administrationto a patient. Examples of appropriate aqueous solvents are as follows:water for injection containing 25 mM TRIS, 5 mg/mL phenol and 16 mg/mLglycerol; water for injection containing 2 mg/mL sodium phosphatedibasic, 1.6 mg/mL m-cresol, 0.65 mg/mL phenol, and 16 mg/mL glycerol;and water for injection containing 25 mM TRIS, 5 mg/mL phenol, 0.1 Mtrisodium citrate, and 16 mg/mL glycerol.

[0298] In a preferred embodiment, the crystals are prepared in a mannerthat obviates the need to separate the crystals from the mother liquor.Thus, it is preferred that the mother liquor itself be suitable foradministration to the patient, or that the mother liquor can be madesuitable for administration by dilution with a suitable diluent. Theterm diluent will be understood to mean a solution comprised of anaqueous solvent in which is dissolved various pharmaceuticallyacceptable excipients, including without limitation, a buffer, anisotonicity agent, zinc, a preservative, protamine, and the like.

[0299] In addition to the derivatized insulin, divalent cation,complexing compound, and hexamer-stabilizing compound, pharmaceuticalcompositions adapted for parenteral administration in accordance withthe present invention may employ additional excipients and carriers suchas water miscible organic solvents such as glycerol, sesame oil, aqueouspropylene glycol and the like. When present, such agents are usuallyused in an amount ranging from about 0.5% to about 2.0% by weight basedupon the final formulation. Examples of such pharmaceutical compositionsinclude sterile, isotonic, aqueous saline solutions of the derivatizedinsulin derivative buffered with a pharmaceutically acceptable bufferand pyrogen free. For further information on the variety of techniquesusing conventional excipients or carriers for parenteral products,please see Remington's Pharmaceutical Sciences, 17th Edition, MackPublishing Company, Easton, Pa., USA (1985), which is incorporatedherein by reference.

[0300] In the broad practice of the present invention, it is alsocontemplated that a formulation may contain a mixture of themicrocrystalline formulation and a soluble fraction of the derivatizedinsulin or a soluble fraction of normal insulin or rapid-acting insulinanalog, such as, LysB28,ProB29-human insulin. Such mixtures are designedto provide a combination of meal-time control of glucose levels, whichis provided by the soluble insulin, and basal control of glucose levels,which is provided by the insoluble insulin.

[0301] The following preparations and examples illustrate and explainthe invention. The scope of the invention is not limited to thesepreparations and examples. Reference to “parts” for solids means partsby weight. Reference to “parts” for liquids means parts by volume.Percentages, when used to express concentration, mean mass per volume(×100). All temperatures are degrees Centigrade (° C.). “TRIS” refers to2-amino-2-hydroxymethyl-1,3,-propanediol. The 1000 part-per-million(ppm) zinc solution was prepared by diluting 1.00 mL of a 10,000 ppmzinc atomic absorption standard solution [Ricca Chemical Company, zincin dilute nitric acid] with water to a final volume of 10.00 mL.

[0302] In many of the preparations described below, the yield ofprecipitates and crystals was estimated. The yield estimate relied ondetermination of the amount of derivatized insulin or derivatizedinsulin analog in the precipitate or crystal, and on an estimate of theamount of the same initially in solution. To determine the amount ofderivatized protein, samples of re-dissolved precipitate or crystal, andof the supernatant above the precipitate or crystals, were analyzed byreversed-phase gradient HPLC, as described below.

[0303] Briefly, the analytical system relied on a C8 reversed-phasecolumn, at 23° C. The flow rate was 1.0 mL/min and UV detection at 214nm was used. Solvent A was 0.1% (vol:vol) trifluroacetic acid in 10:90(vol:vol) acetonitrile:water. Solvent B was 0.1% (vol:vol)trifluroacetic acid in 90:10 (vol:vol) acetonitrile:water. Thedevelopment program was (minutes, % B): (0.1, 0); (45.1, 75); (50.1,100); (55, 100); (57, 0); (72, 0). All changes were linear. Otheranalytical systems could be devised by the skilled person to achieve thesame objective.

[0304] To prepare for the HPLC analysis, aliquots of the well-mixedsuspensions were dissolved by diluting with either 0.01 N HCl or 0.03 NHCl. HPLC analysis of these solutions gave the total mg/mL ofderivatized protein. Aliquots of the suspensions were centrifuged forapproximately 5 minutes in an Eppendorf 5415C microcentrifuge at 14,000rpm. The decanted supernatant was diluted with either 0.01 N or 0.1 NHCl and analyzed by HPLC. The precipitate was washed by re-suspending inDulbecco's phosphate buffered saline (without calcium or magnesium) andre-pelleted by centrifugation. The buffer was decanted and the solid wasre-dissolved in 0.01 N HCl. The re-dissolved precipitate was analyzed byHPLC.

[0305] HPLC was used to confirm the presence of the expected proteins inthe acidified suspension, re-dissolved precipitate, and supernatant andalso to determine protein concentrations. The retention times of peaksin the chromatograms of the re-dissolved precipitates were compared withthe retention times observed for protamine and the insulin compoundsused to make the formulations. The agreement between retention times wasalways good, showing that the protamine and the derivatized proteinsused to make the formulations were actually incorporated in thecrystals. Concentrations of derivatized proteins were determined bycomparing the appropriate peak areas to the areas of a standard.Protamine concentrations were not quantitated. A 0.22 mg/mL solution ofderivatized insulin was used as the standard. A standard containingprotamine was run, but only for the purpose of determining the retentiontime. Protamine concentration was not quantitated.

[0306] In many of the preparations described below, a standardspectrophotometric assay was used to determine how rapidly the crystalsdissolved in Dulbecco's phosphate buffered saline (pH 7.4) at roomtemperature. Significant deviations from the procedure describedimmediately below are noted where appropriate in the descriptions of thepreparations. A spectrophotometer suitable for measuring in theultraviolet range, and equipped with a 1 cm cuvette and a magneticcuvette stirrer was used for all the dissolution assays. The cuvette,containing a small stir bar and 3.00 mL of phosphate buffered saline(PBS), was put into the cell compartment of the spectrophotometer. Theinstrument was set to 320 nm and zeroed against the same buffer. Then4.0 microliters of a well suspended formulation, usually having a totalconcentration approximately equivalent to a U50 formulation, or about1.6 to 1.8 mg/mL, was added to the cuvette. After waiting 1.0 minute formixing, the optical density at 320 nm was recorded. Since the proteinsinvolved in this work do not absorb light at 320 nm, the decrease inoptical density was due to reduction in light scattering as the crystalsdissolved. The time for the optical density to drop to half of itsinitial value is typically reported (t1/2). As a control, 2.0microliters of U100 Humulin® N (i.e., human insulin NPH, which is alsoknown as human NPH insulin) was added to 3.00 mL of PBS buffer, and theoptical density at 320 nm monitored as above. The dissolution half-time(t1/2) for the Humulin® N formulation was about 6 minutes.

PREPARATION 1 Gly(A21), Arg(B31), Arg(B32)-Human Insulin Analog

[0307] Gly(A21)Arg(B31)Arg(B32)-human insulin was obtained from an E.coli fermentation in which a Gly(A21)-human proinsulin precursormolecule was overexpressed into inclusion bodies. A portion (94.7 g) ofinclusion bodies was solubilized in 500 mL of 6 M guanidinehydrochloride containing 0.1 M TRIS, 0.27 M sodium sulfite, and 0.1 Msodium tetrathionate, pH 10.5 at room temperature. The pH was quicklylowered to 8.8 with 12 N HCl. After vigorously stirring in an opencontainer for 45 minutes the pH was lowered to 2.1 with phosphoric acidand the sample centrifuged overnight at 4° C. The supernatant wasdecanted and stored at 4° C. for additional processing. The pellet wasre-extracted with 200 mL of additional pH 10.5 solution (see above) andthen centrifuged for 3 hours at 4° C. This and the previously obtainedsupernatant were each diluted 4× with 100 mM sodium phosphate, pH 4,precipitating the product and other acidic components. After allowingthe precipitate to settle, most of the supernatant was decanted anddiscarded. The resulting suspension was centrifuged, followed bydecanting and discarding of additional supernatant, leaving wet pelletsof the crude Gly(A21)-human proinsulin S-sulfonate precursor. Thepellets were solubilized in 1.5 liters of 7 M deionized urea, adjustingthe pH to 8 with 5 N NaOH and stirring over several hours at 4° C. Salt(NaCl) was then added to achieve 1 M concentration and the sample wasloaded onto a XAD-7 column (14 cm×20 cm, Toso-Haas, Montgomeryville,Pa.), previously flushed with 50% acetonitrile/50% 50 mM ammoniumbicarbonate, 10% acetonitrile/90% 50 mM ammonium bicarbonate, andfinally with 7 M deionized urea/1M NaCl/20 mM TRIS, pH 8. Once loaded,the column was pumped with 4.5 liters of a 7 M deionized urea/1 MNaCl/20 mM TRIS, pH 8 solution, followed by 2.8 liters of 50 mM ammoniumbicarbonate/1 M NaCl, and 6.5 liters of 50 mM ammonium bicarbonate. Thecolumn was eluted with a linear gradient of acetonitrile in 50 mMammonium bicarbonate, while monitoring the eluant by UV at 280 nm. Thepeak of interest, partially purified Gly(A21)-human proinsulinS-sulfonate precursor, was collected, lyophilized, and subjected to afolding/disulfide bond procedure as follows. A quantity (5.4 g) of theprecursor was dissolved in 3 liters of 20 mM glycine, pH 10.5, 4° C.Then, 15 mL of 240 mM cysteine HCl were added with stirring, whilemaintaining the pH at 10.5 and the temperature at 4° C. The reactionsolution was stirred gently at 4° C. for 27 hours and then quenched bylowering the pH to 3.1 with phosphoric acid. Acetonitrile (155 mL) wasadded, and the solution was then loaded onto a 5×25 cm C4 reversed-phasecolumn previously pumped with 60% acetonitrile/40% water/0.1% TFA andequilibrated in 10% acetonitrile/90% water/0.1% TFA. Once loaded thecolumn was pumped with 1 liter of 17.5% acetonitrile/82.5% water/0.1%TFA, then eluted with a linear gradient of acetonitrile in 0.1% TFAwhile monitoring at 280 nm. Selected fractions were pooled andlyophilized with a recovery of 714 mg. For conversion of the proinsulinprecursor to the desired insulin analog, 697 mg of the Gly(A21) humanproinsulin precursor were dissolved in 70 mL 50 mM ammonium bicarbonate,then chilled to 4° C., pH 8.3. A volume (0.14 mL) of a 1 mg/mL solutionof pork trypsin (Sigma Chemical Company, St. Louis, Mo.) in 0.01 N HClwas added to the sample solution which was stirred gently at 4° C. forabout 24 hours. An additional 0.14 mL of the trypsin solution was addedto the reaction solution which was then stirred for an additional 21hours, 45 minutes. The reaction was quenched by lowering the pH to 3.2with 0.7 mL glacial acetic acid and 0.3 mL phosphoric acid. The quenchedGly(A21)Arg(B31)Arg(B32)-human insulin sample solution from the trypticcleavage reaction was diluted 4× with 30% acetonitrile/70% 50 mM aceticacid, pH 3.1, and loaded onto a 1×30 cm S HyperD F (Biosepra,Marlborough, Mass.) cation exchange column previously pumped with 30%acetonitrile/70% 50 mM acetic acid/500 mM NaCl, pH 3.3, and equilibratedin 30% acetonitrile/70% 50 mM acetic acid. Once loaded the column waspumped with about 50 mL of 30% acetonitrile/70% 50 mM acetic acid, theneluted with a linear gradient of NaCl in 30% acetonitrile/50 mM aceticacid while monitoring the eluant at 276 nm. Selected fractionscontaining the Gly(A21)Arg(B31)Arg(B32)-human insulin were pooled,diluted 3× with purified water and loaded onto a 2.2×25 cm C4reversed-phase column (Vydac, Hesperia, Calif.) previously pumped with60% acetonitrile/40% water/0.1% TFA, then 10% acetonitrile/90%water/0.1% TFA. Once loaded, the column was pumped with about 200 mL of10% acetonitrile/90% water/0.1% TFA, then eluted with a linear gradientof acetonitrile in 0.1% TFA. Selected fractions were pooled andlyophilized giving a recovery of 101 mg. Analytical HPLC revealed apurity of greater that 95% main peak. Electrospray mass spectroscopy(ESMS) analysis of the purified protein yielded a molecular weight of6062.9 (6063.0, theory).

PREPARATION 2 Des(B30)-Human Insulin

[0308] Des(B30)-human insulin was prepared from human proinsulin bycontrolled tryptic hydrolysis. A mass (2 g) of human proinsulinbiosynthesized in recombinant E. coli and purified by conventionalmethods [Frank, B. H., et al., in PEPTIDES:Synthesis-Structure-Function. Proceedings of the Seventh AmericanPeptide Symposium, Rich, D. H. and Gross, E. (Eds.), Pierce ChemicalCompany, Rockford, pp. 729-738, 1981; also, Frank, B. H., U.S. Pat. No.4,430,266, issued Feb. 7, 1984, each of which is incorporated byreference] were dissolved in 400 mL of 0.1 M, pH 7.5 HEPES buffer. Afteraddition of 8 mL of 1 M CaCl₂ (in water) and pH adjustment to 7.5 with 5N NaOH, 2 mL of a 10 mg/mL solution of pork trypsin (Sigma) in 0.01 NHCl were transferred to the sample solution while gently stirring. Thereaction solution was allowed to stir at ambient temperature for 2 hoursand 42 minutes, at which time it was transferred to a 37° C. environmentwhile stirring occasionally. After 1 hour and 45 minutes at 37° C. theenzymatic reaction was quenched by lowering the pH to 3.0 withphosphoric acid and the temperature to 4° C. for storage. Subsequently,the solution was brought to room temperature and diluted with 50 mLacetonitrile, then to a final volume of 500 mL with purified water, thenloaded onto a 2.5×58 cm CG-161 (Toso-Haas) column previously pumped with1 c.v. (column volume) of 40% acetonitrile/60% 0.1 M ammonium sulfate,pH 2.5, and 2 c.v. of 10% acetonitrile/90% 0.1 M ammonium sulfate, pH2.5. Once loaded the column was pumped with 1 c.v. of 10%acetonitrile/90% 0.1 M ammonium sulfate, pH 2.5. The column was elutedwith a linear gradient of acetonitrile in 0.1 M ammonium sulfate, pH2.5, while monitoring the eluant at 276 nm. The peak of interest,partially purified des(B30)-human insulin, was collected by poolingselected fractions. This pooled sample of partially purifieddes(B30)-human insulin was diluted to 1.28 liters with purified water,pH 3.5, and applied to a 1×29 cm S HyperD F (Biosepra) cation exchangecolumn previously pumped with 1 c.v. of 30% acetonitrile/70% 0.1%TFA/0.5 M NaCl, pH 1.9, and 2 c.v. of 30% acetonitrile/70% 0.1% TFA, pH2.3. Once loaded the column was pumped with 1 c.v. 30% acetonitrile/70%0.1% TFA, pH 2.3, then eluted with a linear gradient of NaCl in 30%acetonitrile/70% 0.1% TFA, pH 1.9 to 2.3, while monitoring the eluant at276 nm. Selected fractions containing the purified des(B30)-humaninsulin were pooled, diluted 2.5× with purified water and loaded onto a35-c.c. C8 SepPak (Waters, Milford, Mass.) previously cleaned and primedwith 2 c.v. of acetonitrile, 2 c.v. of 60% acetonitrile/40% 0.1% TFA,and 2 c.v. of 10% acetonitrile/90% 0.1% TFA. Once loaded the SepPak wasflushed with 3 c.v. of 10% acetonitrile/90% 0.1% TFA and then elutedwith 2 c.v. of 60% acetonitrile/40% 0.1% TFA. The lyophilized eluantyielded 500 mg. An analytical HPLC assay suggested greater than 95% mainpeak. Electrospray mass spectroscopy (ESMS) analysis of the purifiedprotein yielded a molecular weight of 5706.5 (5707, theory).

PREPARATION 3 Rabbit Insulin

[0309] Rabbit insulin was prepared as described in Chance, R. E., et al.[Proinsulin, Insulin, C-Peptide, Baba, S., et al. (Eds.), ExcerptaMedica, Amsterdam-Oxford, pp. 99-105 (1979)].

PREPARATION 4 Asp(B28)-Human Insulin Analog

[0310] Asp(B28)-human insulin was prepared and purified essentiallyaccording to the teaching of examples, 31 and 32 of Chance, R. E., etal. (U.S. Pat. No. 5,700,662, issued Dec. 23, 1997) which is expresslyincorporated herein by reference. Des(B23-30)-human insulin [Bromer, W.W. and Chance, R. E., Biochim. Biophys. Acta, 133:219-223 (1967), whichis incorporated herein by reference] and a synthetic octapeptideGly-Phe-Phe-Tyr-Thr-Asp-Lys(Tfa)-Thr were condensed usingtrypsin-assisted semisynthesis, purified by gel filtration andreversed-phased HPLC, treated with 15% ammonium hydroxide (v/v) for fourhours at ambient temperature to remove the trifluoroacetate (Tfa)blocking group from Lys(B29), purified by reversed-phase HPLC, andlyophilized.

PREPARATION 5 Syntheses of Derivatized Proteins

[0311] The following is an outline of the syntheses of certainderivatized proteins used to prepare the precipitates and crystals ofthe present invention. The outline is to be read together with the datain Table 2, below.

[0312] A measured mass of purified insulin or of an insulin analog wasdissolved in a measured volume of dimethylsulfoxide (DMSO) withstirring. Then, a measured volume of tetramethylguanidine hydrochloride(TMG) was added and the solution mixed thoroughly. In a separatecontainer, a measured mass of an N-acyl-succinimide (NAS) was dissolvedin a measured volume of DMSO. A measured volume of the second solutionwas added to the first solution. The reaction was carried out at roomtemperature, and the progress of the reaction was monitored by analyzingsamples of the reaction mixture using HPLC. The reaction was quenched byadding a measured volume of ethanolamine, and then acidifying to pH 2-3.

[0313] The reaction mixture was then subjected to purification usingreversed-phase chromatography alone, or using a combination of cationexchange chromatography followed by reversed-phase chromatography. Thereversed-phase purification was carried out using an FPLC® system(Pharmacia) with UV detection at 214 nm or at 280 nm, a fractioncollector, 2.2×25 cm or 5×30 cm C18 column, 2.5 or 5 mL/min flow rate,at room temperature. The liquid phases were mixtures of Solution A [0.1%trifluroacetic acid (TFA) in 10:90 acetonitrile:water (vol:vol)] andSolution B [0.1% trifluroacetic acid (TFA) in 70:30 acetonitrile:water(vol:vol)] appropriate to elute and separate the species of interest.Typically, the column was equilibrated and loaded while in 100% SolutionA. Then, a linear gradient to some proportion of Solution B was used toseparate the reaction products adequately. Fractions containing productwere pooled. The development of purification methods is within the skillof the art.

[0314] Table 2 below provides experimental data, according to theoutline above, for the synthesis of the derivatized proteins that wereused to prepare various embodiments of the present invention. Thestarting proteins were prepared as described above, or according toconventional methods. Conventional purification was used to providehighly purified starting proteins for the syntheses described below. Thesynthesis of insulin, insulin analogs, and proinsulin is within theskill of the art, and may be accomplished using recombinant expression,semisythesis, or solid phase synthesis followed by chain combination.The purification of synthesized proteins to a purity adequate to preparethe derivatives used in the present invention is carried out byconventional purification techniques.

[0315] Molecular weight of the purified derivatives was confirmed bymass spectrometry via electrospray mass analysis (ESMS). Assignment ofthe acylation site was based either on a chromatographic analysis(“HPLC”), or on an N-terminal analysis (“N-terminal”), or both. TABLE 2Summary of synthesis of various derivatized proteins. Starting proteinhuman insulin human insulin human insulin protein mass (mg) 141.3 1,080120 DMSO (mL) 42 30 36 TMG (μL) 30.5 233 25.9 NAS acyl chain n-hexanoyln-octanoyl n-dodecanoyl Mass of NAS (mg) 7.76 85.7 9.22 Volume of DMSO1.0 1.0 0.701 (mL) Volume of NAS 0.494 0.785 0.701 solution added (mL)Reaction time (min) 40 105 40 Ethanolamine volume 20 100 120 (μL) Totalyield (%) 40 33 36 Mol. Wt. (theory) 5906.0 5933.9 5990.0 Mol. Wt.(ESMS) 5906.8 5933.9 5990.0 HPLC Purity (%) 96 94 98 Acylation site NεNε Nε (HPLC) Acylation site Nε Nε Nε (N-terminal) protein mass (mg) 1942040 2050 DMSO (mL) 60 62 58 TMG (μL) 41.9 441 443 NAS acyl chain n-n-butyryl n-hexanoyl tetradecanoyl Mass of NAS (mg) 23.4 269.3 209Volume of DMSO 1.0 1.0 2.0 (mL) Volume of NAS 0.756 0.29 1.44 solutionadded (mL) Reaction time (min) 20 30 30 Ethanolamine volume 5 100 100(μL) Total yield (%) 45 27* 22 Mol. Wt. (theory) 6018.1 5877.8 5905.9Mol. Wt. (ESMS) 6018.2 5877.8 5906.0 HPLC Purity (%) 98 94 93 Acylationsite Nε Nε Nε (HPLC) Acylation site Nε — — (N-terminal)

[0316] The following is an outline of the synthesis of additionalderivatized proteins. The outline is to be read together with the datain Table 3, below, to provide full synthetic schemes.

[0317] A measured mass of purified insulin or of an insulin analog wasdissolved by adding to it a measured volume of 50 mM boric acid, pH2.57. A measured volume of acetonitrile, equal to the volume of boricacid solution, was then added slowly with stirring. The “solvent” volumeis the sum of the volumes of the boric acid and acetonitrile. The pH ofthe solution was adjusted to between 10.2 and 10.5 using NaOH. In aseparate container, a measured mass of an N-acyl-succinimide (“NAS”) wasdissolved in a measured volume of DMSO. A measured volume of the secondsolution was added to the first solution. The reaction was carried outat room temperature, the pH was maintained above 10.2 as necessary, andthe progress of the reaction was monitored by analyzing samples of thereaction mixture using HPLC. The reaction was quenched by acidifying topH 2-3. The reaction mixture was then subjected to purification using areversed-phase chromatography system as described above.

[0318] Table 3 provides experimental data, according to the outlineabove, for the synthesis of the derivatized proteins that were used toprepare various embodiments of the present invention. Molecular weightof the purified derivatives was confirmed by mass spectrometry viaelectrospray mass analysis (ESMS). Assignment of the acylation site wasbased either on a chromatographic analysis (“HPLC”), or on an N-terminalanalysis (“N-terminal”), or both. TABLE 3 Summary of synthesis ofvarious derivatized proteins. Starting protein human insulin humaninsulin human insulin protein mass (mg) 2,170 2,240 2,250 solvent (mL)200 240 200 NAS acyl chain n-butyryl n-pentanoyl n-octanoyl Mass ofN-acyl- 108.7 1155 173 succinimide (mg) Volume of DMSO 1.0 5 1.0 (mL)Volume of NAS 0.955 0.719 0.81 solution added (mL) Reaction time (min)40 40 40 Total yield (%) 25 12 37 Mol. Wt. (theory) 5877.8 5891.8 5933.9Mol. Wt. (ESMS) 5877.7 5891.9 5933.8 HPLC Purity (%) 96 95 96 Acylationsite Nε Nε Nε (HPLC) protein mass (mg) 1,960 2,750 1,040 solvent (mL)200 200 200 NAS acyl chain n-nonanoyl n-dodecanoyl n- tetradecanoyl Massof N-acyl- 145.8 19.9 102.3 succinimide (mg) Volume of DMSO 1.0 1.0 1.0(mL) Volume of NAS 0.887 0.771 0.885 solution added (mL) Reaction time(min) 40 30 35 Total yield (%) 35 14 39 Mol. Wt. (theory) 5947.9 5990.06018.1 Mol. Wt. (ESMS) 5948.1 5989.9 6018.1 HPLC Purity (%) 94 93 94Acylation site Nε Nε Nε (HPLC) sheep Starting protein insulin beefinsulin pork insulin protein mass 312 275 200 solvent (mL) 100 100 100NAS acyl chain n-hexanoyl n-hexanoyl n-octanoyl Mass of N-acyl- 27.219.9 16.4 succinimide (mg) Volume of DMSO 1.0 1.0 1.0 (mL) Volume of NAS0.644 0.771 0.764 solution added (mL) Reaction time (min) 45 30 82 Totalyield (%) 31 50 41 Mol. Wt. (theory) 5801.7 5831.8 5903.9 Mol. Wt. (ms)5801.8 5831.7 5903.9 HPLC Purity (%) 96 96 96 Acylation site Nε Nε Nε(HPLC) rabbit des (B30)- AspB28-human Starting protein insulin humaninsulin insulin Protein mass (mg) 211.4 205.3 132.3 Solvent (mL) 100 2020 NAS acyl chain n-octanoyl n-octanoyl n-octanoyl Mass of N-acyl- 16.821.5 11.5 succinimide (mg) Volume of DMSO 1.0 0.5 1.0 (mL) Volume of NAS0.786 0.303 0.715 solution added (mL) Reaction time (min) 57 40 85 Totalyield (%) 39 47 32 Mol. Wt. (theory) 5919.9 5833.6 5951.9 Mol. Wt. (ms)5920.0 5832.7 5952.2 HPLC Purity (%) 95 96 94 Acylation site Nε Nε Nε(HPLC) GlyA21, ArgB31, ArgB32-human des(B27)- insulin human insulinStarting protein analog human insulin analog Protein mass (mg) 86.2134.8 44.8 Solvent (mL) 10 20 7 NAS acyl chain n-octanoyl 2-methyl-n-octanoyl hexanoyl Mass of N-acyl- 22.4 749 3.6 succinimide (mg) Volumeof DMSO 0.5 4.93* 1.0 (mL) Volume of NAS 0.115 0.052 0.993 solutionadded (mL) Reaction time (min) 40 45 40 Total yield (%) 45 45 53 Mol.Wt. (theory) 6189.2 5919.9 5832.8 Mol. Wt. (ms) 6189.2 5919.9 5832.9HPLC Purity (%) 97 96 93 Acylation site Nε Nε Nε (HPLC) Starting proteinhuman insulin human insulin human insulin Protein mass (mg) 160 147.72,080 Solvent (mL) 20 20 200 NAS acyl chain 4-methyl- 3-methyl-n-octanoyl octanoyl decanoyl Mass of N-acyl- 715 22.5 146.7 succinimide(mg) Volume of DMSO 4.97* 1.0* 1.0* (mL) Volume of NAS 0.0734 0.6090.884 solution added (mL) Reaction time (min) 60 45 40 Total yield (%)54 38 5.3** Mol. Wt. (theory) 5947.9 5976.0 6060.1 Mol. Wt. (ms)****5947.8 5976.2 6060.5 HPLC Purity (%) 96 96 92 Acylation site Nε NεA1-Nα, Nε (HPLC) des(B30)- human insulin Starting protein analog humaninsulin human insulin Protein mass (mg) 205.3 1,960 2,110 Solvent (mL)20 200 200 NAS acyl chain n-octanoyl n-nonanoyl n-decanoyl Mass ofN-acyl- 21.5 145.8 150.5 succinimide (mg) Volume of DMSO 1.0 1.0 1.0(mL) Volume of NAS 0.089 0.0887 0.975 solution added (mL) Reaction time(min) 40 40 60 Total yield (%) 11.0 11.5 11.1 Mol. Wt. (theory) 5959.56088.2 6116.2 Mol. Wt. (ms) 5959.3 6088.3 6116.4 HPLC Purity (%) 96 9292 Acylation site A1-Nα, Nε A1-Nα, Nε A1-Nα, Nε (HPLC)

PREPARATION 6 Microcrystals of B29-Nε-octanoyl-LysB29 Human Insulin

[0319] A dry powder of B29-Nε-octanoyl-LysB29 human insulin (7 parts bymass) is dissolved in 1000 parts by volume of an aqueous solventcomposed of 25 mM TRIS, 0.1 M trisodium citrate, and 10 mg/mL phenol atpH 7.6. To this solution is added 150 parts of a 15.3 mM solution ofzinc chloride. The pH is adjusted to 7.6 with 1 N HCl and/or 1 N NaOH.Then 120 parts by volume of ethanol are added. This solution is filteredthrough a 0.22 micron, low-protein binding filter. A second solution isprepared by dissolving 6 parts by mass of protamine sulfate in 10,000parts by volume of water then filtering through a 0.22 micron,low-protein binding filter. Equal volumes of the acylated insulinsolution and of the protamine sulfate solution are combined. Anamorphous precipitate forms. This suspension is stirred slowly for 24hours at room temperature (typically about 22° C.). The amorphousprecipitate converts to a microcrystalline solid.

PREPARATION 7 Formulation of Microcrystals of B29-Nε-octanoyl-LysB29Human Insulin

[0320] The microcrystals prepared by the method of Preparation 6 areseparated from the mother liquor and are recovered by conventionalsolid/liquid separation methods, such as, filtration, centrifugation, ordecantation. The recovered microcrystals are then suspended in asolution consisting of 25 mM TRIS, 5 mg/mL phenol, and 16 mg/mLglycerol, pH 7.4, so that the final concentration of acylated insulincorresponds to the equivalent of a 100 U/mL solution of insulin.

PREPARATION 8 Microcrystals of B29-Nε-hexanoyl-LysB29 Human Insulin

[0321] A dry powder of B29-Nε-hexanoyl-LysB29 human insulin (7 parts bymass) is dissolved in 1000 parts by volume of an aqueous solventcomposed of 25 mM TRIS, 0.1 M trisodium citrate, 10 mg/mL phenol, and 16mg/mL glycerol at pH 7.6. To this solution is added 150 parts of a 15.3mM solution of zinc chloride. The pH is adjusted to 7.6 with 1 N HCland/or 1 N NaOH. Then 120 parts by volume of ethanol are added. Thissolution is filtered through a 0.22 micron, low-protein binding filter.A second solution is prepared by dissolving 6 parts by mass of protaminesulfate in 10,000 parts by volume of water then filtering through a 0.22micron, low-protein binding filter. Equal volumes of the acylatedinsulin solution and of the protamine sulfate solution are combined. Anamorphous precipitate forms. This suspension is stirred slowly for 24hours at room temperature (typically about 22° C.). The amorphousprecipitate converts to a microcrystalline solid.

PREPARATION 9 Formulation of Microcrystals of B29-Nε-hexanoyl-LysB29Human Insulin

[0322] The microcrystals prepared by the method of Preparation 8 areseparated from the mother liquor and are recovered by conventionalsolid/liquid separation methods. The recovered microcrystals are thensuspended in a solution consisting of 2 mg/mL sodium phosphate dibasic,1.6 mg/mL m-cresol, 0.65 mg/mL phenol, and 16 mg/mL glycerol, pH 6.8, sothat the final concentration of acylated insulin correspondsapproximately to the concentration equivalent of a 100 U/mL solution ofinsulin.

PREPARATION 10 Microcrystals of B28-Nε-octanoyl-LysB28,ProB29-HumanInsulin

[0323] A dry powder of B28-Nε-octanoyl-LysB28-LysB28,ProB29-humaninsulin (7 parts by mass) is dissolved in 1000 parts by volume of anaqueous solvent composed of 25 mM TRIS, 0.1 M trisodium citrate, and 10mg/mL phenol at pH 7.6. To this solution is added 150 parts of a 15.3 mMsolution of zinc chloride. The pH is adjusted to 7.6 with 1 N HCl and/or1 N NaOH. Then 120 parts by volume of ethanol are added. This solutionis filtered through a 0.22 micron, low-protein binding filter. A secondsolution is prepared by dissolving 6 parts by mass of protamine sulfatein 10,000 parts by volume of water then filtering through a 0.22 micron,low-protein binding filter. Equal volumes of the acylated insulinsolution and of the protamine sulfate solution are combined. Anamorphous precipitate forms. After 24 hours at room temperature(typically about 22° C.), the amorphous precipitate converts to amicrocrystalline solid.

PREPARATION 11 Formulation of Microcrystals ofB28-Nε-octanoyl-LysB28,ProB29-Human Insulin

[0324] The microcrystals prepared by the method of Preparation 10 areseparated from the mother liquor and are recovered by conventionalsolid/liquid separation methods. The recovered microcrystals are thensuspended in a solution consisting of 25 mM TRIS, 5 mg/mL phenol, 0.1 Mtrisodium citrate, and 16 mg/mL glycerol, pH 7.6, so that the finalconcentration of acylated insulin corresponds approximately to theconcentration equivalent of a 100 U/mL solution of insulin.

PREPARATION 12 Microcrystals of B28-Nε-butyryl-LysB28,ProB29-HumanInsulin

[0325] A dry powder of B29-Nε-butyryl-LysB29 human insulin (7 parts bymass) is dissolved in 1000 parts by volume of an aqueous solventcomposed of 25 mM TRIS, 0.1 M trisodium citrate, and 10 mg/mL phenol atpH 7.6. To this solution is added 150 parts of a 15.3 mM solution ofzinc chloride. The pH is adjusted to 7.6 with 1 N HCl and/or 1 N NaOH.Then 120 parts by volume of ethanol are added. This solution is filteredthrough a 0.22 micron, low-protein binding filter. A second solution isprepared by dissolving 6 parts by mass of protamine sulfate in 10,000parts by volume of water then filtering through a 0.22 micron,low-protein binding filter. Equal volumes of the acylated insulinsolution and of the protamine sulfate solution are combined. Anamorphous precipitate forms. After 24 hours at room temperature(typically about 22° C.), the amorphous precipitate converts to amicrocrystalline solid.

PREPARATION 13 Formulation of Microcrystals ofB28-Nε-butyryl-LysB28,ProB29-Human Insulin

[0326] The microcrystals prepared by the method of Preparation 7 areseparated from the mother liquor and are recovered by conventionalsolid/liquid separation methods. The recovered microcrystals are thensuspended in a solution consisting of 25 mM TRIS, 2.5 mg/mL m-cresol,and 16 mg/mL glycerol, pH 7.8, so that the final concentration ofacylated insulin corresponds approximately to the concentrationequivalent of a 100 U/mL solution of insulin.

PREPARATION 14 Microcrystals of B29-Nε-butyryl-Human Insulin

[0327] B29-Nε-butyryl-human insulin was prepared as described inPreparation 5. A sample of B29-Nε-butyryl-human insulin (16.21 mg) wasdissolved in 0.8 mL of 0.1 N HCl. After stirring for 5-10 minutes, avolume (0.32 mL) of a zinc nitrate solution containing 1000parts-per-million (ppm) zinc(II) was added, and the resulting solutionwas thoroughly mixed by stirring. Then, 3.2 mL of crystallizationdiluent was added, and the resulting mixture was stirred untilcompletely mixed. The crystallization diluent was prepared by dissolvingin water, with stirring, 0.603 g of TRIS, 1.007 g of phenol, 1.582 g ofglycerol and 2.947 g of trisodium citrate. Further water was added tobring the of the solution to 100 mL. After mixing the zinc-insulinderivative solution with the crystallization diluent, the pH of theresulting solution was adjusted to 7.59 using small aliquots of 1 N HCland 1 N NaOH, as needed. The pH-adjusted solution was filtered through a0.22 micron, low-protein binding filter. To a volume of the filteredsolution was added an equal volume of an aqueous solution of protamine,prepared by dissolving 18.59 mg of protamine sulfate in water to a finalvolume of 50 mL. The mixture of the two volumes was swirled gently tocomplete mixing, and then allowed to stand at 25° C. Rod-like crystalsformed in very high yield (greater than 90%). In the spectrophotometricdissolution assay described above, the crystals of B29-Nε-butyryl-humaninsulin had a t1/2 of 32-33 minutes, compared with about 6 minutes forHumulin® N in the same assay.

PREPARATION 15 Microcrystals of B29-Nε-pentanoyl-Human Insulin

[0328] B29-Nε-pentanoyl-human insulin was prepared as described inPreparation 5. A sample of B29-Nε-pentanoyl-human insulin (16.14 mg) wasdissolved in 0.8 mL of 0.1 N HCl. The procedure described in Preparation14 was followed, except the pH was adjusted to 7.60, and the protaminesolution contained 18.64 mg of protamine sulfate in a total volume of 50mL. Rod-like crystals formed in high yield (greater than 80%). In thespectrophotometric dissolution assay described above, the crystals ofB29-Nε-pentanoyl-human insulin had a t1/2 of 33-34 minutes, comparedwith about 6 minutes for Humulin® N in the same assay.

PREPARATION 16 Microcrystals of B29-Nε-hexanoyl-Human Insulin

[0329] B29-Nε-hexanoyl-human insulin was prepared as described inPreparation 5. A sample of B29-Nε-hexanoyl-human insulin (15.87 mg) wasdissolved in 0.8 mL of 0.1 N HCl. The procedure described in Preparation14 was followed, except the pH was adjusted to 7.58. Rod-like crystalsformed in very high yield (greater than 90%). In the spectrophotometricdissolution assay described above, the crystals of B29-Nε-hexanoyl-humaninsulin had a t1/2 of 69-70 minutes, compared with about 6 minutes forHumulin® N in the same assay.

PREPARATION 17 Microcrystals of B29-Nε-(2-methylhexanoyl)-Human Insulin

[0330] B29-Nε-(2-methylhexanoyl)-human insulin was prepared as describedin Preparation 5. A mass (8.19 mg) of B29-Nε-(2-methylhexanoyl)-humaninsulin was dissolved in 0.400 mL of 0.1 N HCl. After stirring for 5-10minutes, 0.160 mL of a zinc nitrate solution containing 1000parts-per-million (ppm) zinc(II) was added and the resulting solutionwas mixed thoroughly. Then, 1.60 mL of diluent (in 100 mL: 0.604 g TRIS,1.003 g phenol, 3.218 g glycerol, and 3.069 g trisodium citrate) wasadded and mixed. The pH of this solution was adjusted to 7.61 with smallquantities of 1.0 N HCl and 1.0 N NaOH, and then the solution wasfiltered through a 0.22 micron, low-protein binding filter. To 2 mL ofthis filtered solution was added 2 mL of a protamine solution (in 100mL, 37.41 mg of protamine). The mixture was swirled gently. Aprecipitate formed. The mixture was left undisturbed at 25° C. Rod-likecrystals formed in good yield (greater than 65%). In thespectrophotometric dissolution assay described above, the crystals ofB29-Nε-2-methylhexanoyl-human insulin had a t1/2 of 23 minutes, comparedwith about 6 minutes for Humulin® N in the same assay.

PREPARATION 18 Microcrystals of B29-Nε-octanoyl-Human Insulin

[0331] B29-Nε-octanoyl-LysB29 human insulin (4.17 mg) was dissolved in 1mL of a solvent composed of 25 mM TRIS, 0.1 M trisodium citrate, and 10mg/mL phenol at pH 7.6. To this solution, 0.15 mL of a 15.3 mM solutionof zinc chloride was added. The resulting solution was adjusted to a pHof 7.6 with 1 N NaOH. To this solution 0.12 mL of ethanol was added. Theresulting solution was filtered through a 0.22 micron, low-proteinbinding filter. A second solution was prepared by dissolving 3.23 mg ofprotamine sulfate in 10 mL of water then filtered through a 0.22 micron,low-protein binding filter. A volume of 1 mL of theB29-Nε-octanoyl-LysB29 human insulin solution and 1 mL of the protaminesulfate solution were combined, resulting in the immediate appearance ofan amorphous precipitate. This solution was divided into two 1 mLportions which were transferred to vials then gently agitated for 19hours at room temperature (approximately 22° C.), using an automaticwrist-action shaker. This procedure resulted in the formation of awhite-to-off-white microcrystalline solid. HPLC analysis of crystalsthat have been removed from the mother liquor and thoroughly washeddemonstrated the presence of protamine within the crystalline material.

PREPARATION 19 Crystalline Suspension Formulation ComprisingNε-octanoyl-Human Insulin

[0332] An acidic solution of Nε-octanoyl-human insulin was prepared bydissolving 39.7 mg of Nε-octanoyl-human insulin in 1 mL of 0.1 N HCl.This solution was stirred for approximately 5 minutes. To this solutionwas added 0.4 mL of a zinc nitrate solution containing 1000parts-per-million (ppm) zinc(II) with stirring. The zinc nitratesolution was a 1:10 dilution of a 10,000 ppm Zn(II) atomic absorptionstandard. To the Nε-octanoyl-human insulin plus zinc solution was added4 mL of a crystallization diluent (40 mg/mL glycerol, 50 mM TRIS, 4mg/mL m-cresol, 1.625 mg/mL phenol, 100 mM trisodium citrate, pH 7.4).The pH of the resulting solution was adjusted to 7.61. The pH-adjustedsolution was filtered through a 0.22 micron, low protein-binding filter.Five milliliters (5 mL) of protamine solution (37.6 mg of protaminesulfate in 50 mL of water) were added to 5 mL of the filteredNε-octanoyl-human insulin solution. The solution was allowed to standundisturbed for 63 hours at a controlled temperature of 25° C.

[0333] Microscopic inspection (at 63 hours) revealed thatcrystallization had occurred and that the preparation had yieldeduniform, single, rod-like crystals possessing approximate averagelengths of about 10 microns.

[0334] The crystals were sedimented by allowing the formulation to standundisturbed. Eight milliliters (8 mL) of the supernatant were thenremoved, and were replaced with 8 mL of a diluent [16 mg/mL glycerol, 20mM TRIS, 1.6 mg/mL m-cresol, 0.65 mg/mL phenol, 40 mM trisodium citrate,pH 7.4]. The crystals were then resuspended. This procedure was carriedout in the same way three times, except that on the third occasion, the8 mL of supernatant was replaced with 7 mL of diluent.

[0335] The amount of insulins in the formulation was analyzed by HPLC toquantitate the total potency. The total potency refers to the totalconcentration of human insulin and Nε-hexanoyl-human insulin. An aliquot(0.050 mL) of the fully resuspended formulation was dissolved in 0.950mL of 0.01 N HCL, and subjected to HPLC analysis, as described below.For HPLC analyses, the following conditions were used: aC8-reversed-phase column; constant 23° C.; 1.0 mL/min, detection at 214nm; solvent A=10% acetonitrile (vol/vol) in 0.1% aqueous trifluoroaceticacid; solvent B=90% acetonitrile (vol/vol) in 0.1% aqueoustrifluoroacetic acid; linear gradients (0.1 min, 0% B; 45.1 min, 75% B;50.1 min, 100% B; 55 min 100% B; 57 min, 0% B; 72 min, 0% B). Standardswere prepared by dissolving bulk insulin and bulk acyl insulin in 0.01 NHCl. The concentration of each standard was determined by UVspectroscopy. A solution of 1.000 mg/mL of human insulin in a 1 cmcuvette was assumed to have an absorbance of 1.05 optical density unitsat the wavelength maximum (approximately 276 nm). This corresponds to amolar extinction coefficient of 6098. Acylated insulins were assumed tohave the same molar extinction coefficient as human insulin. Thesolutions calibrated by UV were then diluted to get standards at 0.220,0.147, 0.073, and 0.022 mg/mL. The standards were run on HPLC and astandard curve of area vs. concentration was obtained.

[0336] Total potency of Nε-octanoyl-human insulin in the crystalformulation was 3.76 mg/mL. The concentration of solubleNε-octanoyl-human insulin was determined to be 0.01 mg/mL. No unacylatedhuman insulin was found by HPLC analysis.

[0337] The dissolution rate of the crystals was measured by placing0.005 mL of the uniformly suspended formulation into 3 mL of Dulbecco'sphosphate buffered saline (without calcium or magnesium) in a 1 cm pathlength square quartz cuvette at a temperature of 22° C. This solutionwas stirred at a constant rate using a magnetic cuvette stirrer.Absorbance measurements at 320 nm were taken at 1 minute intervals. Theabsorbance at 320 nm corresponds to the light scattered by the insolubleparticles present in the aqueous suspension. Consequently, as themicrocrystals dissolve, the absorbance approaches zero. The approximatetime required for the 0.005 mL of this formulation to dissolve was morethan 400 minutes. The time required for a 0.005 mL sample of U100commercial Humulin® N to dissolve under the same conditions was about 10minutes.

[0338] Particle size measurement was performed on a sample of theformulation utilizing a particle sizing instrument (Multisizer ModelIIE, Coulter Corp., Miami, Fla. 33116-9015). To perform thismeasurement, 0.25 mL of the crystal formulation was added to 100 mL of adiluent consisting 14 mM dibasic sodium phosphate, 16 mM glycerol, 1.6mg/mL m-cresol, 0.65 mg/mL phenol, pH 7.4. The instrument aperture tubeorifice size was 50 microns. Particle size data were collected for 50seconds. The mean particle diameter of the crystals was approximately 6microns, with an approximately normal distribution, encompassing a rangeof particle sizes from approximately 2 microns to approximately 12microns. This result is similar to the particle size distribution ofcommercial NPH as reported in DeFelippis, M. R., et al., J. Pharmaceut.Sci. 87:170-176 (1998).

PREPARATION 20 Microcrystals of B29-Nε-nonanoyl-Human Insulin

[0339] Nε-nonanoyl-human insulin was prepared as described inPreparation 5. A sample of Nε-nonanoyl-human insulin (16.16 mg) wasdissolved in 0.8 mL of 0.1 N HCl. The procedure described in Preparation14 was followed, except the protamine solution contained 18.64 mg ofprotamine sulfate in a total volume of 50 mL. Rod-like crystals formedin high yield (greater than 80%). In the spectrophotometric dissolutionassay described above, the crystals of B29-Nε-nonanoyl-human insulin hada t1/2 of 83 minutes, compared with about 6 minutes for Humulin® N inthe same assay.

PREPARATION 21 Microcrystals of B29-Nε-decanoyl-Human Insulin

[0340] B29-Nε-decanoyl-human insulin was prepared essentially asdescribed in Preparation 5. A sample of B29-Nε-decanoyl-human insulin(60.7 mg) was dissolved in 1.5 mL of 0.1 N HCl. A volume (0.6 mL) of azinc nitrate solution containing 1000 parts-per-million (ppm) zinc(II)was added and mixed thoroughly. To 0.7 mL of the resulting solution in“Vial A” was added 2.0 mL of diluent “A” (50 mM citrate, 5 mg/mL phenol,16 mg/mL glycerol, 25 mM TRIS, pH 7.6). To another 0.7 mL portion of thezinc-derivatized protein solution was added 2.0 mL of diluent “B” (100mM citrate, 2 mg/mL phenol, 50 mM TRIS, pH 7.6). The pH in the vials wasadjusted to 7.62 and 7.61, respectively, and each was filtered through a0.22 micron, low-protein binding filter. A volume of the contents ofvial A (2.5 mL) was mixed with 2.5 mL of a protamine sulfate solution(7.4 mg protamine sulfate dissolved in 10 mL of diluent A). A cloudyprecipitate developed immediately. The preparation was allowed to standundisturbed at 25° C. Likewise, a volume of the contents of vial B (2.5mL) was mixed with 2.5 mL of a protamine sulfate solution (37.8 mgprotamine sulfate dissolved in 50 mL of water). A cloudy precipitatedeveloped immediately. The preparation was allowed to stand undisturbedat 25° C. Microscopic examination of the contents of both vials after 60hours revealed that small crystals had formed in both. A rod-likemorphology was clearly evident for the crystals in vial B. The yield ofcrystals in vial B was determined by HPLC to be 80%. In thespectrophotometric dissolution assay described above, the crystals ofB29-Nε-decanoyl-human insulin in vial B had a t1/2 of 70 minutes,compared with about 6 minutes for Humulin® N in the same assay.

PREPARATION 22 Microcrystals of B29-Nε-dodecanoyl-Human Insulin

[0341] B29-Nε-dodecanoyl-human insulin was prepared as described inPreparation 5. A sample of B29-Nε-dodecanoyl-human insulin (17.00 mg)was dissolved in 4.0 mL of diluent (containing in an aqueous solution,per mL of solution, 10 mg phenol, 32 mg glycerol, 30 mg trisodiumcitrate dihydrate, and 6.1 mg TRIS, pH 8.47). The pH of the solution ofthe insulin analog derivative was adjusted to 8.57 using small aliquotsof 1 N NaOH. To the pH-adjusted solution was added 0.320 mL of a zincnitrate solution containing 1000 parts-per-million (ppm) zinc(II). ThepH of the zinc-insulin analog derivative solution was adjusted to 7.59using small aliquots of 1 N HCl and 1 N NaOH. The pH-adjusted solutionwas filtered through a 0.22 micron, low-protein binding filter. To 2.0mL of the resulting solution in “Vial A” was added 0.25 mL of ethanol.The mixture was mixed gently. To another 2.0 mL volume of the resultingsolution in “Vial B” was added 0.6 mL of ethanol. The mixture was mixedgently. To the contents of both Vial A and Vial B were added 2.0 mL of aprotamine solution (containing, dissolved in water, 0.376 mg protamineper mL). After adding the protamine solution, each vial contained acloudy suspension. Each vial was swirled gently to complete mixing, andthen allowed to stand at 25° C. Small, irregular crystals formed in veryhigh yield (greater than 90%). In the spectrophotometric dissolutionassay described above, the crystals of B29-Nε-dodecanoyl-human insulinhad a t1/2 of greater than 300 minutes, compared with about 6 minutesfor Humulin® N in the same assay.

PREPARATION 23 Microcrystals of B29-Nε-tetradecanoyl-Human Insulin

[0342] B29-Nε-tetradecanoyl-human insulin was prepared as described inPreparation 5. A sample of B29-Nε-tetradecanoyl-human insulin (16.42 mg)was dissolved in 0.5 mL of 0.1 N HCl. After stirring for 5-10 minutes, avolume (0.32 mL) of a zinc nitrate solution containing 1000parts-per-million (ppm) zinc(II) was added, and the resulting solutionwas thoroughly mixed by stirring. Then, 3.2 mL of diluent (containing inan aqueous solution, per mL of solution, 10 mg phenol, 32 mg glycerol,30 mg trisodium citrate dihydrate, and 6.1 mg TRIS, pH 7.58), and theresulting mixture was stirred until completely mixed. After mixing thezinc-insulin derivative solution with the diluent, the pH of theresulting solution was adjusted first to 7.9, and then back to 7.59,using small aliquots of 1 N HCl and 1 N NaOH, as needed. The pH-adjustedsolution was filtered through a 0.22 micron, low-protein binding filter.To 1.97 mL of the resulting solution in “Vial A” was added 0.246 mL ofethanol. The mixture was mixed gently. To another 1.97 mL volume of theresulting solution in “Vial B” were added 0.591 mL of ethanol, whichresulted in the formation of a haziness in the vial. The mixture wasmixed gently. To the contents of both Vial A and Vial B were added 1.97mL of a protamine solution (containing, dissolved in water, 0.376 mgprotamine per mL). After adding the protamine solution, each vialcontained a cloudy suspension. Each vial was swirled gently to completemixing, and then allowed to stand at 25° C. Small, irregular crystalsformed in very high yield (greater than 90%). In the spectrophotometricdissolution assay described above, the crystals ofB29-Nε-tetradecanoyl-human insulin had a t1/2 of greater than 300minutes, compared with about 6 minutes for Humulin® N in the same assay.

PREPARATION 24 Microcrystals of B29-Nε-hexadecanoyl-Human Insulin

[0343] B29-Nε-hexadecanoyl-human insulin was prepared as described inPreparation 5. A sample of B29-Nε-hexadecanoyl-human insulin (16.29 mg)was dissolved in 0.5 mL of 0.1 N HCl. After stirring for 5-10 minutes, avolume (0.32 mL) of a zinc nitrate solution containing 1000parts-per-million (ppm) zinc(II) was added, and the resulting solutionwas thoroughly mixed by stirring. Then, 3.2 mL of diluent (containing inan aqueous solution, per mL of solution, 10 mg phenol, 32 mg glycerol,30 mg trisodium citrate dihydrate, and 6.1 mg TRIS, pH 7.58), and theresulting mixture was stirred until completely mixed. After mixing thezinc-insulin derivative solution with the diluent, the pH of theresulting solution was adjusted first to 8.0, and then back to 7.61,using small aliquots of 1 N HCl and 1 N NaOH, as needed. The pH-adjustedsolution was filtered through a 0.22 micron, low-protein binding filter.To 2.0 mL of the resulting solution in “Vial A” was added 0.25 mL ofethanol. The mixture was mixed gently. To another 2.0 mL volume of theresulting solution in “Vial B” were added 0.6 mL of ethanol, whichresulted in the formation of a haziness in the vial. The mixture wasmixed gently. To the contents of both Vial A and Vial B were added 2.0mL of a protamine solution (containing, dissolved in water, 0.376 mgprotamine per mL). After adding the protamine solution, each vialcontained a cloudy suspension. Each vial was swirled gently to completemixing, and then allowed to stand at 25° C. Crystals formed in bothvials. Small, irregular crystals formed in very high yield (greater than90%). In the spectrophotometric dissolution assay described above, thecrystals of B29-Nε-hexadecanoyl-human insulin had a t1/2 of greater than300 minutes, compared with about 6 minutes for Humulin® N in the sameassay.

PREPARATION 25 Microcrystals of A1-Nα-octanoyl-B29-Nε-octanoyl-HumanInsulin

[0344] A mass (8.13 mg) of A1-Nα-octanoyl-B29-Nε-octanoyl-human insulinanalog was dissolved in 1.60 mL of diluent (in 100 mL: 0.604 g TRIS,1.003 g phenol, 3.218 g glycerol, and 3.069 g trisodium citrate). The pHof this solution was adjusted to 7.61 with small quantities of 1.0 N HCland 1.0 N NaOH. After stirring for 5-10 minutes, 0.160 mL of a zincnitrate solution containing 1000 parts-per-million (ppm) zinc(II) wasadded and the resulting solution was mixed again thoroughly. The pH wasadjusted again, to 7.62, and then the solution was filtered through a0.22 micron, low-protein binding filter. To 2 mL of this filteredsolution was added 2 mL of a protamine solution (in 100 mL, 37.41 mg ofprotamine). The mixture was swirled gently. A precipitate formed. Themixture was left undisturbed at 25° C. Small irregular crystals formed.

PREPARATION 26 Microcrystals ofA1-Nα-octanoyl-B29-Nε-octanoyl-desB30-Human Insulin

[0345] The process of Preparation 25 was followed essentially, exceptthat 8.08 mg of A1-Nα-octanoyl-B29-Nε-octanoyl-desB30-human insulin wasused. Small irregular crystals formed.

PREPARATION 27 Microcrystals ofA1-Nα-nonanoyl-B29-Nε-nonanoyl-desB30-Human Insulin

[0346] The process of Preparation 25 was followed essentially, exceptthat 8.07 mg of A1-Nα-nonanoyl-B29-Nε-nonanoyl-desB30-human insulin wasused. Small irregular crystals formed.

PREPARATION 28 Microcrystals ofA1-Nα-decanoyl-B29-Nε-decanoyl-desB30-Human Insulin

[0347] The process of Preparation 25 was followed essentially, exceptthat 8.22 mg of A1-Nα-decanoyl-B29-Nε-decanoyl-desB30-human insulin wasused. Small irregular crystals formed.

PREPARATION 29 Microcrystals ofB29-Nε-octanoyl-Gly(A21),Arg(B31),Arg(B32)-Human Insulin Analog

[0348] B29-Nε-octanoyl-Gly(A21),Arg(B31),Arg(B32) -human insulin analogwas prepared as described in Preparation 5. A mass (8.6 mg) ofB29-Nε-octanoyl-Gly(A21),Arg(B31),Arg(B32)-human insulin analog wasdissolved in 0.4 mL of 0.1 N HCl. After stirring for 5-10 minutes, 0.160mL of a zinc nitrate solution containing 1000 parts-per-million (ppm)zinc(II) was added and the resulting solution was mixed againthoroughly. Then, 1.60 mL of diluent (in 100 mL: 0.604 g TRIS, 1.003 gphenol, 3.218 g glycerol, and 3.069 g trisodium citrate) was added andmixed by additional stirring. The pH of this solution was adjusted to7.59 with small quantities of 1.0 N HCl and 1.0 N NaOH, and then thesolution was filtered through a 0.22 micron, low-protein binding filter.To 2 mL of this filtered solution was added 2 mL of a protamine solution(in 100 mL, 37.41 mg of protamine). The mixture was swirled gently. Aprecipitate formed. The mixture was left undisturbed at 25° C. Smallirregular crystals formed.

PREPARATION 30 Microcrystals of B29-Nε-octanoyl-des(ThrB30)-HumanInsulin

[0349] B29-Nε-octanoyl-des(ThrB30)-human insulin was prepared asdescribed in Preparation 5. A sample ofB29-Nε-octanoyl-des(ThrB30)-human insulin (16.21 mg) was dissolved in0.5 mL of 0.1 N HCl. After stirring for 5-10 minutes, a volume (0.32 mL)of a zinc nitrate solution containing 1000 parts-per-million (ppm)zinc(II) was added, and the resulting solution was thoroughly mixed bystirring. Then, 3.2 mL of diluent (containing in an aqueous solution,per mL of solution, 10 mg phenol, 32 mg glycerol, 30 mg trisodiumcitrate dihydrate, and 6.1 mg TRIS, pH 7.58), and the resulting mixturewas stirred until completely mixed. After mixing the zinc-insulinderivative solution with the diluent, the pH of the resulting solutionwas adjusted first to 8.45 using small aliquots of 1 N NaOH, as needed.This failed to completely clarify the solution. The pH was then adjustedto 7.61, using small aliquots of 1 N HCl, as needed. The pH-adjustedsolution was filtered through a 0.22 micron, low-protein binding filter.To a volume of the filtered solution was added an equal volume of anaqueous solution of protamine (containing, dissolved in water, 0.376 mgprotamine per mL). After adding the protamine solution, a cloudysuspension developed. The suspension was swirled gently to completemixing, and then allowed to stand at 25° C. Rod-like crystals formed inhigh yield (greater than 80%). In the spectrophotometric dissolutionassay described above, the crystals of B29-Nε-octanoyl-des(ThrB30)-humaninsulin had a t1/2 of 94 minutes, compared with about 6 minutes forHumulin® N in the same assay.

PREPARATION 31 Microcrystals of B29-Nε-octanoyl-des(B30)-Human InsulinAnalog

[0350] B29-Nε-octanoyl-des(ThrB30)-human insulin was prepared asdescribed in Preparation 5. A sample ofB29-Nε-octanoyl-des(ThrB30)-human insulin (8.09 mg) was dissolved in0.400 mL of 0.1 N HCl. After stirring for 5-10 minutes, a volume (0.16mL) of a zinc nitrate solution containing 1000 parts-per-million (ppm)zinc(II) was added, and the resulting solution was thoroughly mixed bystirring. After stirring for 5-10 minutes, 0.160 mL of a zinc nitratesolution containing 1000 parts-per-million (ppm) zinc(II) was added andthe resulting solution was mixed thoroughly. Then, 1.60 mL of diluent(in 100 mL: 0.604 g TRIS, 1.003 g phenol, 3.218 g glycerol, and 3.069 gtrisodium citrate) was added and mixed. The pH of this solution wasadjusted to 7.61 with small quantities of 1.0 N HCl and 1.0 N NaOH, andthen the solution was filtered through a 0.22 micron, low-proteinbinding filter. To 2 mL of this filtered solution was added 2 mL of aprotamine solution (in 100 mL, 37.41 mg of protamine). The mixture wasswirled gently. A precipitate formed. The mixture was left undisturbedat 25° C.

PREPARATION 32 Microcrystals of B29-Nε-hexanoyl-Beef Insulin

[0351] B29-Nε-hexanoyl-beef insulin was prepared as described inPreparation 5. A sample of B29-Nε-hexanoyl-beef insulin (16.14 mg) wasdissolved in 0.8 mL of 0.1 N HCl. After stirring for 5-10 minutes, avolume (0.32 mL) of a zinc nitrate solution containing 1000parts-per-million (ppm) zinc(II) was added, and the resulting solutionwas thoroughly mixed by stirring. Then, 3.2 mL of crystallizationdiluent (containing, per mL, 10 mg phenol, 16 glycerol, 30 mg trisodiumcitrate dihydrate, and 6.0 mg TRIS, in water, pH unadjusted) was added,and the resulting mixture was stirred until completely mixed. Aftermixing the zinc-insulin derivative solution with the crystallizationdiluent, the pH of the resulting solution was adjusted to 7.58 usingsmall aliquots of 1 N HCl and 1 N NaOH, as needed. The pH-adjustedsolution was filtered through a 0.22 micron, low-protein binding filter.To a volume of the filtered solution was added an equal volume of anaqueous solution of protamine (0.375 mg protamine sulfate/mL solution,in water, pH not adjusted). The mixture of the two volumes was swirledgently to complete mixing. A cloudy suspension formed, which was gentlyswirled to complete mixing, and then allowed to stand at 25° C. Rod-likecrystals formed in very high yield (greater than 90%). In thespectrophotometric dissolution assay described above, the crystals ofB29-Nε-hexanoyl-beef insulin had a t1/2 of greater than 300 minutes,compared with about 6 minutes for Humulin® N in the same assay.

PREPARATION 33 Microcrystals of B29-Nε-hexanoyl-Sheep Insulin

[0352] B29-N^(ε)-hexanoyl-sheep insulin was prepared as described inPreparation 5. A sample of B29-Nε-hexanoyl-sheep insulin (16.15 mg) wasdissolved in 0.8 mL of 0.1 N HCl. After stirring for 5-10 minutes, avolume (0.32 mL) of a zinc nitrate solution containing 1000parts-per-million (ppm) zinc(II) was added, and the resulting solutionwas thoroughly mixed by stirring. The solution was hazy, and theaddition of 0.1 mL 0.1 N HCl did not cause the haze to dispersecompletely. The procedure of Preparation 32 was followed thereafter,except that the pH was adjusted to 7.61 instead of 7.58. Small,irregular crystals formed in high yield (greater than 80%). In thespectrophotometric dissolution assay described above, the crystals ofB29-Nε-hexanoyl-sheep insulin had a t1/2 of 184 minutes, compared withabout 6 minutes for Humulin® N in the same assay.

PREPARATION 34 Microcrystals of B29-Nε-octanoyl-Pork Insulin

[0353] B29-Nε-octanoyl-pork insulin was prepared as described inPreparation 5. A sample of B29-Nε-octanoyl-pork insulin (16.78 mg) wasdissolved in 0.5 mL of 0.1 N HCl. After stirring for 5-10 minutes, avolume (0.32 mL) of a zinc nitrate solution containing 1000parts-per-million (ppm) zinc(II) was added, and the resulting solutionwas thoroughly mixed by stirring. Then, 3.2 mL of diluent (containing inan aqueous solution, per mL of solution, 10 mg phenol, 32 mg glycerol,30 mg trisodium citrate dihydrate, and 6.1 mg TRIS, pH 8.5), and theresulting mixture was stirred until completely mixed. After mixing thezinc-insulin derivative solution with the diluent, the pH of theresulting solution was adjusted first to 8.4 using small aliquots of 1 NNaOH, as needed. The pH was then adjusted to 7.60, using small aliquotsof 1 N HCl, as needed. After this, the procedure of Preparation 32 wasfollowed. Small, irregular crystals formed in high yield (greater than80%). In the spectrophotometric dissolution assay described above, thecrystals of B29-Nε-octanoyl-pork insulin had a t1/2 of greater than 300minutes, compared with about 6 minutes for Humulin® N in the same assay.

PREPARATION 35 Microcrystals of B29-Nε-octanoyl-Rabbit Insulin

[0354] B29-Nε-octanoyl-rabbit insulin was prepared as described inPreparation 5. A sample of B29-Nε-octanoyl-rabbit insulin (8.10 mg) wasdissolved in 0.25 mL of 0.1 N HCl. After stirring for 5-10 minutes, avolume (0.16 mL) of a zinc nitrate solution containing 1000parts-per-million (ppm) zinc(II) was added, and the resulting solutionwas thoroughly mixed by stirring. Then, 1.6 mL of diluent (containing inan aqueous solution, per mL of solution, 10 mg phenol, 32 mg glycerol,30 mg trisodium citrate dihydrate, and 6.1 mg TRIS, pH 8.5), and theresulting mixture was stirred until completely mixed. After mixing thezinc-insulin derivative solution with the diluent, the pH of theresulting solution was adjusted first to 8.34 using small aliquots of 1N NaOH, as needed. The pH was then adjusted to 7.57, using smallaliquots of 1 N HCl, as needed. After this, the procedure of Preparation32 was followed. Small, irregular crystals formed in high yield (greaterthan 80%). In the spectrophotometric dissolution assay described above,the crystals of B29-Nε-octanoyl-rabbit insulin had a t1/2 of 119minutes, compared with about 6 minutes for Humulin® N in the same assay.

PREPARATION 36 Microcrystals of B29-Nε-octanoyl-des(B27)-Human InsulinAnalog

[0355] B29-Nε-octanoyl-des(B27)-human insulin analog was prepared asdescribed in Preparation 5. A mass (8.02 mg) ofB29-Nε-octanoyl-des(B27)-human insulin analog was dissolved in 0.400 mLof 0.1 N HCl. After stirring for 5-10 minutes, 0.160 mL of a zincnitrate solution containing 1000 parts-per-million (ppm) zinc(II) wasadded and the resulting solution was mixed thoroughly. Then, 1.60 mL ofdiluent (in 100 mL: 0.604 g TRIS, 1.003 g phenol, 3.218 g glycerol, and3.069 g trisodium citrate) was added and mixed. The pH of this solutionwas adjusted to 7.61 with small quantities of 1.0 N HCl and 1.0 N NaOH,and then the solution was filtered through a 0.22 micron, low-proteinbinding filter. To 2 mL of this filtered solution was added 2 mL of aprotamine solution (in 100 mL, 37.41 mg of protamine). The mixture wasswirled gently. A precipitate formed. The mixture was left undisturbedat 25° C. After six days, well-formed, single, rod-shaped crystalsformed.

PREPARATION 37 Microcrystals of B29-N^(ε)-octanoyl-Asp(B28)-HumanInsulin Analog

[0356] A mass (8.16 mg) of B29-Nε-octanoyl-Asp(B28)-human insulin analogwas dissolved in 1.60 mL of diluent (in 100 mL: 0.604 g TRIS, 1.003 gphenol, 3.218 g glycerol, and 3.069 g trisodium citrate). The pH of thissolution was adjusted to 7.61 with small quantities of 1.0 N HCl and 1.0N NaOH. After stirring for 5-10 minutes, 0.160 mL of a zinc nitratesolution containing 1000 parts-per-million (ppm) zinc(II) was added andthe resulting solution was mixed again thoroughly. The pH was adjustedagain, to 7.62, and then the solution was filtered through a 0.22micron, low-protein binding filter. To 2 mL of this filtered solutionwas added 2 mL of a protamine solution (in 100 mL, 37.41 mg ofprotamine). The mixture was swirled gently. A precipitate formed. Themixture was left undisturbed at 25° C. Small, irregular crystals formedin high yield (greater than 80%). In the spectrophotometric dissolutionassay described above, the crystals of B29-N^(ε)-octanoyl-Asp(B28)-humaninsulin analog had a t1/2 of 15 minutes, compared with about 6 minutesfor Humulin® N in the same assay.

PREPARATION 38 Microcrystals of B28-Nε-butryl-LysB28,ProB29-HumanInsulin

[0357] B28-Nε-butryl-LysB28,ProB29-human insulin was prepared asdescribed in Preparation 5. A sample ofB28-Nε-butryl-LysB28,ProB29-human insulin (16.09 mg) was dissolved in0.8 mL of 0.1 N HCl. After stirring for 5-10 minutes, a volume (0.32 mL)of a zinc nitrate solution containing 1000 parts-per-million (ppm)zinc(II) was added, and the resulting solution was thoroughly mixed bystirring. Then, 3.2 mL of crystallization diluent was added, and theresulting mixture was stirred until completely mixed. (Thecrystallization diluent was prepared by dissolving in water, withstirring, 0.603 g of TRIS, 1.007 g of phenol, 1.582 g of glycerol and2.947 g of trisodium citrate. Further water was added to bring the ofthe solution to 100 mL.) After mixing the zinc-insulin derivativesolution with the crystallization diluent, the pH of the resultingsolution was adjusted to 7.60 using small aliquots of 1 N HCl and 1 NNaOH, as needed. The pH-adjusted solution was filtered through a 0.22micron, low-protein binding filter. To a volume of the filtered solutionwas added an equal volume of an aqueous solution of protamine, preparedby dissolving 18.64 mg of protamine sulfate in water to a final volumeof 50 mL. The mixture of the two volumes was swirled gently to completemixing, and then allowed to stand at 25° C.

PREPARATION 39 Microcrystals of B28-Nε-hexanoyl-LysB28,ProB29-HumanInsulin

[0358] B28-Nε-hexanoyl-LysB28,ProB29-human insulin was prepared asdescribed in Preparation 5. A sample ofB28-Nε-hexanoyl-LysB28,ProB29-human insulin (15.95 mg) was dissolved in0.8 mL of 0.1 N HCl. The procedure of Preparation 38 was subsequentlyfollowed. Small, irregular crystals formed in high yield (greater than80%). In the spectrophotometric dissolution assay described above, thecrystals of B28-Nε-hexanoyl-LysB28,ProB29-human insulin had a t1/2 of5-6 minutes, compared with about 6 minutes for Humulin® N in the sameassay.

PREPARATION 40 Microcrystals of B28-Nε-hexanoyl-LysB28,ProB29-HumanInsulin

[0359] B28-Nε-hexanoyl-LysB28,ProB29-human insulin was prepared asdescribed in Preparation 5. A sample ofB28-Nε-hexanoyl-LysB28,ProB29-human insulin (16.8 mg) was dissolved in4.0 mL of diluent (containing in an aqueous solution, per mL ofsolution, 10 mg phenol, 32 mg glycerol, 30 mg trisodium citratedihydrate, and 6.1 mg TRIS, pH 7.58). The pH of the solution of theinsulin analog derivative was adjusted to 8.4 using small aliquots of 1N NaOH. To the pH-adjusted solution was added 0.320 mL of a zinc nitratesolution containing 1000 parts-per-million (ppm) zinc(II). The pH of thezinc-insulin analog derivative solution was adjusted to 7.61 using smallaliquots of 1 N HCl and 1 N NaOH. The pH-adjusted solution was filteredthrough a 0.22 micron, low-protein binding filter. To 2.0 mL of theresulting solution in “Vial A” was added 0.25 mL of ethanol. The mixturewas mixed gently, and the solution became hazy. To another 2.0 mL volumeof the resulting solution in “Vial B” was added 0.6 mL of ethanol. Themixture was mixed gently, and solution became hazy. To the contents ofboth Vial A and Vial B were added 2.0 mL of a protamine solution(containing, dissolved in water, 0.376 mg protamine per mL). Afteradding the protamine solution, each vial contained a cloudy suspension.Each vial was swirled gently to complete mixing, and then allowed tostand at 25° C. Crystals formed in both vials. The composition of thesolution in Vial A was analyzed for the remaining concentration ofinsulin analog derivative, and the crystals were subjected todissolution testing.

PREPARATION 41 Microcrystals of B28-Nε-octanoyl-LysB28,ProB29-HumanInsulin

[0360] B28-Nε-octanoyl-LysB28,ProB29-human insulin was prepared asdescribed in Preparation 5. A sample ofB28-Nε-octanoyl-LysB28,ProB29-human insulin (16.02 mg) was dissolved in0.8 mL of 0.1 N HCl. Hereafter, the procedure of Preparation 38 wasfollowed essentially. Small, irregular crystals formed in high yield(greater than 80%). In the spectrophotometric dissolution assaydescribed above, the crystals of B28-Nε-octanoyl-LysB28,ProB29-humaninsulin had a t1/2 of 7-8 minutes, compared with about 6 minutes forHumulin® N in the same assay.

PREPARATION 42 Microcrystals of B28-Nε-octanoyl-LysB28,ProB29-HumanInsulin

[0361] B28-Nε-octanoyl-LysB28,ProB29-human insulin was prepared asdescribed in Preparation 5. A sample ofB28-Nε-octanoyl-LysB28,ProB29-human insulin (16.35 mg) was dissolved in4.0 mL of diluent (containing in an aqueous solution, per mL ofsolution, 10 mg phenol, 32 mg glycerol, 30 mg trisodium citratedihydrate, and 6.1 mg TRIS, pH 7.58). The pH of the solution of theinsulin analog derivative was adjusted to 8.4 using small aliquots of 1N NaOH. To the pH-adjusted solution was added 0.320 mL of a zinc nitratesolution containing 1000 parts-per-million (ppm) zinc(II). The pH of thezinc-insulin analog derivative solution was adjusted to 7.62 using smallaliquots of 1 N HCl and 1 N NaOH. The pH-adjusted solution was filteredthrough a 0.22 micron, low-protein binding filter. To 2.0 mL of theresulting solution in “Vial A” was added 0.25 mL of ethanol. The mixturewas mixed gently, and the solution became hazy. To another 2.0 mL volumeof the resulting solution in “Vial B” was added 0.6 mL of ethanol. Themixture was mixed gently, and solution became hazy. To the contents ofboth Vial A and Vial B were added 2.0 mL of a protamine solution(containing, dissolved in water, 0.376 mg protamine per mL). Afteradding the protamine solution, each vial contained a cloudy suspension.Each vial was swirled gently to complete mixing, and then allowed tostand at 25° C. Crystals formed in both vials. The composition of thesolution in Vial A was analyzed for the remaining concentration ofinsulin analog derivative, and the crystals were subjected todissolution testing. Small, irregular crystals formed in high yield(greater than 80%). In the spectrophotometric dissolution assaydescribed above, the crystals of B29-Nε-octanoyl-rabbit insulin had at1/2 of 15 minutes, compared with about 6 minutes for Humulin® N in thesame assay.

PREPARATION 43 Amorphous Suspension of B29-Nε-octanoyl-Human Insulin

[0362] B29-Nε-octanoyl-human insulin was prepared as described inPreparation 5. A sample of B29-Nε-octanoyl-human insulin (20.31 mg ofsolid, containing 16.95 mg protein) was dissolved in 0.5 mL of 0.1 NHCl. Then 200 microliters of a zinc nitrate solution containing 1000parts-per-million (ppm) zinc(II) was added, followed by 2.0 mL of adiluent containing per mL: 1.625 mg phenol, 4 mg m-cresol, 40 mgglycerol, 5 mg anhydrous sodium dibasic phosphate, and 7.5 mg trisodiumcitrate dihydrate, with a final pH of 7.6. After adding the diluent, thepH of the resulting solution was adjusted to 7.58 with 0.090 mL of 1 NNaOH. The solution was then passed through a 0.22 micron,low-protein-binding sterile filter, and refrigerated overnight. At thispoint, the concentration of the insulin derivative was 6.074 mg/mL. Thenext morning, the solution had no visible precipitate. A volume of thesolution (2.50 mL) was mixed with 2.875 mL of a protamine sulfatesolution containing per mL 0.75 mg of solid protamine sulfate in water,and an amorphous precipitate immediately formed. The concentration ofB29-Nε-octanoyl-human insulin was 2.825 mg/mL after adding protamine.The suspension was injected into two dogs approximately 1 hour and fortyminutes after mixing the insulin derivative with protamine.

PREPARATION 44 Amorphous Suspension of B28-Nε-myristoyl-LysB28,ProB29Human Insulin Analog

[0363] B28-Nε-myristoyl-LysB28,ProB29-human insulin was preparedessentially as described in Preparation 5. A sample ofB28-Nε-myristoyl-LysB28,ProB29-human insulin (20.43 mg of solid, 18.53mg of protein) was dissolved in 0.5 mL of 0.1 N HCl. Then 200microliters of a zinc nitrate solution containing 1000 parts-per-million(ppm) zinc(II) and 2.0 mL of formulation diluent were added. Theformulation diluent contained, per mL: 1.6 mg phenol, 4 mg m-cresol, 40mg glycerol, 5 mg anhydrous sodium dibasic phosphate, and 7.5 mgtrisodium phosphate dihydrate, with a final pH of 7.6. The pH of theformulation was adjusted from 5.9 to 8.7 with 100 microliters of 1 NNaOH. The formulation was clear. The pH was then reduced to 7.59 byadding 20 microliters of 1 N HCl. At this point, the proteinconcentration was 6.57 mg/mL. The solution was filtered through a 0.22micron, low-protein binding sterile filter and refrigerated overnight.The next morning, the formulation had no visible precipitate present. Aportion of the solution (2.50 mL) was mixed with 2.875 mL of protaminesolution (0.75 mg/mL of solid protamine sulfate dissolved in water) andan amorphous suspension formed. The concentration ofB28-Nε-myristoyl-LysB28,ProB29-human insulin would have been reduced to3.056 mg/mL by the addition of the protamine solution. Samples for HPLCanalysis were prepared promptly after the protamine was added. Based onknown peak retention times, the HPLC analysis showed that the insolublematerial contained protamine and B28-Nε-myristoyl-LysB28,ProB29-humaninsulin. The concentration of B28-Nε-myristoyl-LysB28,ProB29-humaninsulin in the supernatant was found to be 0.005 mg/mL, and in a sampleof the precipitate re-dissolved to the original volume, theconcentration was 3.13 mg/mL. The concentration ofB28-Nε-myristoyl-LysB28,ProB29-human insulin in a sample of acidifiedsuspension was 3.34 mg/mL.

PREPARATION 45 Microcrystals of B29-Nε-octanoyl-Human Insulin

[0364] A dry powder of B29-Nε-octanoyl-human insulin (7 parts by mass)is dissolved in 175 parts by volume of 0.1 N HCl, and then a solution ofzinc chloride (60 parts by volume, prepared by dissolving zinc oxide inHCl to give a 15.3 mM concentration of zinc) is added. To this solutionis added 800 parts by volume of an aqueous solvent comprising 25 mMTRIS, 10 mg/mL phenol, 0.1 M citrate, 40 mg/mL glycerol, in water at pHvalue 7.6. The resulting solution is adjusted to pH value of 7.6, andthen filtered through a 0.22 micron, low-protein binding filter.

[0365] An additional solution is prepared by dissolving 7 parts by massof protamine sulfate in 10,000 parts by volume of water. The protaminesolution is filtered through a 0.22 micron, low-protein binding filter.Equal volumes of the derivatized protein solution and the protaminesolution are combined by adding the protamine solution to the acylatedinsulin solution. An amorphous precipitate forms. This suspension isallowed to stand undisturbed for 48 hours at a temperature of 25° C. Themicrocrystals in the resulting preparation will provide extended andflatter time action compared with an equal dose of NPH human insulin.

PREPARATION 46 Microcrystals of B29-Nε-octanoyl-Human Insulin

[0366] The process of Preparation 45 is followed. The suspension isallowed to stand undisturbed for 48 hours at a temperature of 30° C.Similar results are obtained.

PREPARATION 47 Microcrystals of B29-Nε-octanoyl-Human Insulin

[0367] A dry powder of B29-Nε-octanoyl-human insulin (7 parts by mass)is dissolved in 175 parts by volume of 0.1 N HCl, and then a solution ofzinc chloride (60 parts by volume, prepared by dissolving zinc oxide inHCl to give a 15.3 mM concentration of zinc) is added. To this solutionis added 1000 parts by mass of an aqueous solvent comprising 35 mMsodium phosphate dibasic, 4 mg/mL m-cresol, 1.6 mg/mL phenol, 25 mMcitrate, and 40 mg/mL glycerol, in water, pH 7.6. The resulting solutionis adjusted to pH 7.6, and then filtered through a 0.22 micron,low-protein binding filter.

[0368] An additional solution is prepared by dissolving 6 parts by massof protamine sulfate in 10,000 parts by volume of water then filteringthrough a 0.22 micron, low-protein binding filter. Equal volumes of thederivatized insulin solution and the protamine solution are combined byadding the protamine solution to the acylated protein solution. Anamorphous precipitate forms. This suspension is allowed to standundisturbed for 1 week at a temperature of 25° C. The microcrystals inthe resulting preparation will provide extended and flatter time actioncompared with an equal dose of NPH human insulin.

PREPARATION 48 Microcrystals of B29-Nε-octanoyl-Human Insulin

[0369] A dry powder of B29-Nε-octanoyl-human insulin (7 parts by mass)is dissolved in 175 parts by volume of 0.1 N HCl, and then a solution ofzinc chloride (60 parts by volume, prepared by dissolving zinc oxide inHCl to give a 15.3 mM concentration of zinc) is added. To this solutionis added 1000 parts by mass of a solvent comprising 35 mM sodiumphosphate dibasic, 4 mg/mL m-cresol, 1.6 mg/mL phenol, 10 mM citrate, 40mg/mL glycerol, in water, pH 7.6. The resulting solution is adjusted topH 7.6, and then filtered through a 0.22 micron, low-protein bindingfilter.

[0370] An additional solution is prepared by dissolving 6 parts by massof protamine sulfate in 10,000 parts by volume of water then filteringthrough a 0.22 micron, low-protein binding filter. Equal volumes of theacylated insulin solution and the protamine solution are combined byadding the protamine solution to the acylated insulin solution. Anamorphous precipitate forms. This suspension is allowed to standundisturbed for 1 week at a temperature of 25° C. The microcrystals inthe resulting preparation will provide extended and flatter time actioncompared with an equal dose of NPH human insulin.

PREPARATION 49 Microcrystals of B29-Nε-octanoyl-Human Insulin

[0371] The process of Preparation 47 is followed. The suspension isallowed to stand undisturbed for 60 hours at a temperature of 30° C. Themicrocrystals in the resulting preparation will provide extended andflatter time action compared with an equal dose of NPH human insulin.

PREPARATION 50 Microcrystals of B29-Nε-octanoyl-Human Insulin

[0372] A solution is prepared by adding to water for injection (WFI,1000 parts by volume): phenol (0.65 parts by mass), m-cresol (1.6 partsby mass) and glycerin (16 parts by mass). Protamine sulfate powder (0.6parts by mass) is then dissolved in this solution. A solution of zincchloride (60 parts by volume) prepared by dissolving zinc oxide in HClto give a 15.3 mM concentration of zinc in 0.1 N HCl is then added. Adry powder of B29-Nε-octanoyl-human insulin (7 parts by mass) is addedand dissolved with stirring. The pH is adjusted to about 3 to aiddissolution if necessary with small quantities of 1 N HCl and 1 N NaOH.The pH is then adjusted to within the range 3-3.6 with small quantitiesof 1 N HCl and 1 N NaOH. This solution is filtered through a 0.22micron, low-protein binding filter.

[0373] A second solution is prepared by dissolving sodium phosphatedibasic (7.56 parts by mass), phenol (0.65 parts by mass), m-cresol (1.6parts by mass) and glycerin (16 parts by mass) in water for injection(1000 parts by volume). The pH of this solution is adjusted to a valuesuch that combination of a volume of this solution with an equal volumeof the B29-Nε-octanoyl-human insulin solution results in a pH value ofabout 7.5 to about 7.7. After appropriately adjusting the pH of thisbuffer solution, it is filtered through a 0.22 micron, low-proteinbinding filter. Equal volumes of the buffer solution and theB29-Nε-octanoyl-human insulin solution are combined. An amorphousprecipitate forms immediately which becomes crystalline upon standingfor 60 hours undisturbed at a controlled temperature of 25° C. Themicrocrystals in the resulting preparation will provide extended andflatter time action compared with an equal dose of NPH human insulin.

PREPARATION 51 Microcrystals of B29-Nε-octanoyl-Human Insulin

[0374] A solution is prepared by adding to water for injection (1000parts by volume) sodium phosphate dibasic (3.78 parts by mass), phenol(0.65 parts by mass), m-cresol (1.6 parts by mass) and glycerin (16parts by mass). A solution of zinc chloride (6 parts by volume) preparedby dissolving zinc oxide in HCl to give a 153 mM concentration of zincin 0.1 N HCl is then added. A dry powder of B29-Nε-octanoyl-humaninsulin (7 parts by mass) is added and dissolved with stirring. The pHis adjusted to about 3 to aid dissolution if necessary with smallquantities of 1 N HCl and 1 N NaOH. The pH is then adjusted to 7.6 with10% HCl and 10% NaOH. This solution is filtered through a 0.22 micron,low-protein binding filter.

[0375] A second solution is prepared by dissolving sodium phosphatedibasic (3.78 parts by mass), phenol (0.65 parts by mass), m-cresol (1.6parts by mass) and glycerin (16 parts by mass) in water for injection(1000 parts by volume). Protamine sulfate powder (0.6 parts by mass) isthen dissolved in this solution. The pH of this solution is adjusted to7.6. This solution is filtered through a 0.22 micron, low-proteinbinding filter. Equal volumes of this protamine solution and theB29-Nε-octanoyl-human insulin solution are combined. An amorphousprecipitate forms immediately which becomes crystalline upon standingfor 60 hours undisturbed at a controlled temperature of 25° C. Themicrocrystals in the resulting preparation will provide extended andflatter time action compared with an equal dose of NPH human insulin.

PREPARATION 52 Microcrystals of B29-Nε-(2-ethylhexanoyl)-Human Insulin

[0376] B29-Nε-(2-ethylhexanoyl)-human insulin was prepared as describedin Preparation 5. A mass (8.00 mg) of B29-Nε-(2-ethylhexanoyl)-humaninsulin was dissolved in 0.400 mL of 0.1 N HCl. Thereafter, theprocedure of Preparation 17 was followed essentially. A precipitateformed. The mixture was left undisturbed at 25° C. Rod-like crystalsformed in high yield (greater than 80%). In the spectrophotometricdissolution assay described above, the crystals ofB29-Nε-2-ethylhexanoyl-human insulin had a t1/2 of 34-35 minutes,compared with about 6 minutes for Humulin® N in the same assay.

EXAMPLE 1 In Vivo Testing in Diabetic Dogs

[0377] The protracted action of a suspension formulation containingmicrocrystals prepared as described in any of Preparations herein istested in diabetic dogs by comparing its ability to controlhyperglycemia with that of control compounds. A one-per-day dose ofabout 0.2 units/kg of body weight is used. This dose would be equivalentto about 1.2 nmol/kg. On test days, blood glucose is monitored for 24hours following subcutaneous injection of the suspension formulation.Control compounds are human insulin and NPH human insulin. Suspensionformulations of microcrystals of the present invention will reduce bloodglucose levels and will have an extended time action compared with humaninsulin NPH when tested at comparable doses.

EXAMPLE 2 Time-Action of Crystals in Rats

[0378] To 1.898 mL of the crystal formulation prepared according toPreparation 19 was added 3.102 mL of a diluent (16 mg/mL glycerol, 20 mMTRIS, 1.6 mg/mL m-cresol, 0.65 mg/mL phenol, 40 mM trisodium citrate, pH7.4). This provided 5 mL of a U40 formulation, which was tested inBBDP/Wor rats, a genetically-characterized animal model, maintained by,and available from, the University of Massachusetts Medical Center(Worchester, Mass.) in connection with Biomedical Research Models, Inc.(Rutland, Mass.). The DPBB/Wor rat line is diabetes-prone, and exhibitsinsulin-dependent (autoimmune) diabetes mellitus.

[0379] Forty BBDP/Wor rats [20 male/20 female, aged 4-5 months,maintained on a long-acting insulin (PZI)], were randomly assigned bygender to eight experimental groups, A, B, C, D, E, F, G, and H. GroupsA (5 males) and B (5 females) were treated for two days with a U40 humaninsulin ultralente composition having 2.5 mg/mL zinc. Groups C (5 males)and D (5 females) were treated for two days with a U40 human insulinultralente composition having 1.25 mg/mL zinc. Groups E (5 males) and F(5 females) were treated for two days with a U40 beef-pork PZI insulin(PZI). Groups G (5 males) and H (5 females) were treated for two dayswith a crystal formulation according to the present invention, asdescribed in this example. Each rat was given daily injections of itsgroup's formulation for the two days before blood glucose wasdetermined, and on the day that the blood glucose was determined.

[0380] Blood was obtained half an hour before administering the testformulations. Animals were injected subcutaneously with either 0.9 U/100g body weight (males) or 1.1 U/100 g body weight (females) at 11:30 A.M.Blood was obtained by nicking the tail (not anaesthetized), storedbriefly on ice, centrifuged, and glucose determined using a Beckman IIglucose analyzer. Blood samples were obtained just prior toadministering the test formulations, and at 2, 4, 6, 8, 12, 16, 20, and24 hours after administration. The crystal formulations of the presentinvention controlled blood glucose for a time comparable to thatobtained with the long-acting insulin preparations.

EXAMPLE 3 Time-Action of Crystals in Rats

[0381] The testing procedure described above in Example 2 was repeatedwith a second 5 mL sample of a U40 formulation of a suspension, preparedas described above.

[0382] Thirty-five BBDP/Wor rats [18 male/17 female, age 4-5 months,maintained on a long-acting insulin (PZI)], were randomly assigned bygender to six experimental groups, I, J, K, L, M, and N. Groups I (8males) and J (8 females) were treated for three days with the crystalformulation according to the present invention, as described in thisexample, above. Groups K (5 males) and L (4 females) were treated forthree days with a U40 human insulin ultralente composition having 2.5mg/mL zinc. Groups M (5 males) and N (5 females) were treated for threedays with a U40 beef-pork PZI insulin (PZI). Each rat was given dailyinjections of its group's formulation for the three days before bloodglucose was determined, and on the day that the blood glucose wasdetermined.

[0383] Blood was obtained half an hour before administering the testformulations. Animals were injected subcutaneously with either 0.9 U/100g body weight (males) or 1.1 U/100 g body weight (females) at 11:30 A.M.Blood was obtained by nicking the tail (not anaesthetized), storedbriefly on ice, centrifuged, and glucose determined using a Beckman IIglucose analyzer. Blood samples were obtained just prior toadministering the test formulations, and at 2, 4, 6, 8, 12, 16, 20, and24 hours after administration. The crystal formulations of the presentinvention controlled blood glucose for a time comparable to thatobtained with the long-acting insulin preparations.

EXAMPLE 4 Time-Action of Crystals in Rats

[0384] Twenty-six BBDP/Wor rats [13 male/13 female, age 4-6 months,maintained on a long-acting, protamine zinc insulin (PZI)], wererandomly assigned by gender to four experimental groups, O, P, Q, and R.Groups O (8 males) and P (8 females) were treated for three days withthe crystal formulation according to the present invention, as describedin Example 2. Groups Q (5 males) and R (5 females) were treated forthree days with a U-40 beef-pork PZI insulin (PZI). Each rat was givendaily injections of its group's formulation for the three days beforeblood glucose was determined, and on the day that the blood glucose wasdetermined.

[0385] Blood was obtained half an hour before administering the testformulations. Animals were injected subcutaneously with either 0.9 U/100g body weight (males) or 1.1 U/100 g body weight (females) at 11:30.Blood was obtained by nicking the tail (not anaesthetized), storedbriefly on ice, centrifuged, and glucose determined using a Beckman IIglucose analyzer. Blood samples were obtained just prior toadministering the test formulations, and at 2, 4, 6, 8, 12, 16, 20, and24 hours after administration. The crystal formulations of the presentinvention controlled blood glucose for a time comparable to thatobtained with the long-acting insulin preparations.

EXAMPLE 5 Amorphous Precipitate of B29-Nε-octanoyl-Human Insulin Testedin Dogs

[0386] The time action of a formulation containing an amorphousprecipitate of protamine and B29-Nε-octanoyl-human insulin, prepared asdescribed in Preparation 43, was determined in two normal dogs (2nmol/kg, subcutaneous). The dogs received a constant infusion ofsomatostatin to create a transient diabetic state. The data werecompared with those observed in the same model after administration ofhuman insulin ultralente (3 nmol/kg, n=5), and with saline (n=6).

[0387] Experiments were conducted in overnight-fasted,chronically-cannulated, conscious male and female beagles weighing 10-17kg (Marshall Farms, North Rose, N.Y.). At least ten days prior to thestudy, animals were anesthetized with isoflurane (Anaquest, Madison,Wis.), and silicone catheters attached to vascular access ports (V-A-P™,Access Technologies, Norfolk Medical, Skokie, Ill.) were inserted intothe femoral artery and femoral vein. The catheters were filled with aglycerol/heparin solution (3:1, v/v; final heparin concentration of 250kIU/mL; glycerol from Sigma Chemical Co., St. Louis, Mo., and heparinfrom Elkins-Sinn, Inc., Cherry Hill, N.J.) to prevent catheterocclusion, and the wounds were closed. Kefzol (Eli Lilly & Co.,Indianapolis, Ind.) was administered pre-operatively (20 mg/kg, IV and20 mg/kg, I.M.), and Keflex was administered post-operatively (250 mg,p.o. once daily for seven days) to prevent infections. Torbugesic (1.5mg/kg, I.M.) was administered post-operatively to control pain.

[0388] Blood was drawn just prior to the study day to determine thehealth of the animal. Only animals with hematocrits above 38% andleukocyte counts below 16,000/mm³ were used (hematology analyzer:Cell-Dyn 900, Sequoia-Turner, Mountain View, Calif.).

[0389] The morning of the experiment, the ports were accessed (AccessTechnologies, Norfolk Medical, Skokie, Ill.); the contents of thecatheters were aspirated; the catheters were flushed with saline (BaxterHealthcare Corp., Deerfield, Ill.); the dog was placed in a cage; andextension lines (protected by a stainless steel tether and attached to aswivel system [Instech Laboratories, Plymouth Meeting, Pa.]) wereattached to the port access lines.

[0390] Dogs were allowed at least 10 minutes to acclimate to the cageenvironment before an arterial blood sample was drawn for determinationof fasting insulin and blood glucose concentrations (time=−30 minutes).At this time, a continuous, IV infusion of cyclic somatostatin (0.65μg/kg/min; BACHEM California, Torrance, Calif.) was initiated andcontinued for the next 30.5 hours. Thirty minutes after the start ofinfusion (time=0 minutes), an arterial blood sample was drawn, and asubcutaneous bolus of test substance, or vehicle, was injected in thedorsal aspect of the neck. Arterial blood samples were taken every 3hours thereafter for the determination of plasma glucose and insulinconcentrations.

[0391] Arterial blood samples were collected in vacuum blood collectiontubes containing disodium EDTA (Terumo Medical Corp., Elkton, Md.) andimmediately placed on ice. The samples were centrifuged, and theresulting plasma was transferred to polypropylene test tubes and storedon ice for the duration of the study.

[0392] Plasma glucose concentrations were determined the day of thestudy using a glucose oxidase method in a Beckman glucose analyzer(Beckman Instruments, Inc., Brea, Calif.). Samples for other assays werestored at −80° C. until time for analysis. Insulin concentrations weredetermined using a double antibody radioimmunoassay.

[0393] At the conclusion of the experiment, the catheters were flushedwith fresh saline, treated with Kefzol (20 mg/kg), and filled with theglycerol/heparin mixture; antibiotic (Keflex; 250 mg) was administeredp.o. To minimize the number of animals being used and to allow pairingof the data base when possible, animals were studied multiple times.Experiments in animals being restudied were carried out a minimum of oneweek apart.

[0394] The formulation of amorphous precipitate of B29-Nε-octanoyl-humaninsulin, prepared as described above, provided effective control ofblood glucose for almost 27 hours, compared with only about 21 hours forhuman insulin ultralente. The precipitate provided a significantlyflatter and a more extended control of glucose levels than did humaninsulin ultralente. For example, the nadir of the blood glucoseconcentration was obtained after 1.5 hours for the precipitate, and thenthe glucose level rose to a fairly constant level. By comparison, thenadir for human insulin ultralente was reached after 9 hours, and afterthat, the blood glucose level rose relatively quickly. The glucose levelat the nadir was 72 mg/dL for the derivatized insulin precipitateformulation, while it was 56 mg/dL for the ultralente formulation.Finally, plasma insulin levels corroborate these observations, andcorrelate well with the greater flatness and extension of time action ofthe amorphous precipitate of B29-Nε-octanoyl-human insulin compared withhuman insulin ultralente.

EXAMPLE 6 Microcrystals of B29-Nε-octanoyl-Human Insulin Tested in Dogs

[0395] The glucodynamics of two formulations containing crystals ofB29-Nε-octanoyl-human insulin, prepared as described in Preparation 19,or essentially as described in Preparation 19 was determined in innormal dogs, using essentially the protocol described in Example 5. Oneof two preparations of microcrystals was administered to each dog at adose of 2 nmol/kg subcutaneously. The experiments were carried out onethree different occasions. The data from these three experiments werecombined. A total of ten dogs each received a 2 nmol/kg dose of one oftwo preparations. In a separate experiment, a dose of a formulation ofmicrocrystals of B29-Nε-octanoyl-human insulin prepared essentially asdescribed in Preparation 19 was administered subcutaneously to each offive dogs at a dose of 3 nmol/kg. Human insulin NPH (2 nmol/kg, n=5),and saline vehicle (n=5) served as controls. In each experiment, thedogs received a constant infusion of somatostatin to create a transientdiabetic state.

[0396] The formulation of microcrystals comprising B29-Nε-octanoyl-humaninsulin, administered at 2 nmol/kg, had an effective time action of 27hours, compared with 21 hours for human insulin NPH at the same dose.The glucodynamic profile showed less hypoglycemic tendency than humaninsulin NPH, which is an advantageous quality. At 3 nmol/kg, themicrocrystals comprising B29-Nε-octanoyl-human insulin effectivelycontrolled glucose levels for at least 30 hours. At the glucose nadir,about the same glucose level was obtained as that obtained afteradministration of human insulin NPH (namely, about 65 mg/dL). However,the duration of such depressed glucose levels was much shorter for themicrocrystals comprising B29-Nε-octanoyl-human insulin (about 3 hours)compared with human insulin NPH (about 7.5 hours). After the nadir,blood glucose levels for the group receiving microcrystals comprisingB29-Nε-octanoyl-human insulin varied only between 91 and 115 mg/dL up to30 hours, after which no further data are available. In contrast,glucose levels in the group the received human insulin NPH at 2 nmol/kgvaried from 89 to 145 after the nadir was reached.

EXAMPLE 7 Microcrystals of B29-Nε-hexanoyl-Human Insulin Tested in Dogs

[0397] The glucodynamics of formulations containing crystals ofB29-Nε-hexanoyl-human insulin, prepared as described in Preparation 16,was determined in normal dogs, using essentially the protocol describedin Example 5. The formulation of microcrystals comprisingB29-Nε-hexanoyl-human insulin, administered at 2 nmol/kg had aneffective time action of 24 hours, compared with 24 hours for humaninsulin NPH at the same dose. The glucodynamic profile was flatter,showing less hypoglycemic tendency than human insulin NPH. At theglucose nadir, about the same glucose level was obtained as thatobtained after administration of human insulin NPH (namely, about 67mg/dL, versus 64 mg/dL for human insulin NPH). However, the duration ofsuch depressed glucose levels was much shorter for the microcrystalscomprising B29-Nε-hexanoyl-human insulin (about 3 hours) compared withhuman insulin NPH (about 7.5 hours).

[0398] The principles, preferred embodiments and modes of operation ofthe present invention have been described in the foregoingspecification. The invention which is intended to be protected herein,however, is not to be construed as limited to the particular formsdisclosed, since they are to be regarded as illustrative rather thanrestrictive. Variations and changes may be made by those skilled in theart without departing from the spirit of the invention.

I claim:
 1. A microcrystal comprising: a) a derivatized protein selectedfrom the group consisting of derivatized insulin, derivatized insulinanalogs, and derivatized proinsulins; b) a complexing compound; c) ahexamer-stabilizing compound; and d) a divalent metal cation.
 2. Themicrocrystal of claim 1 , wherein the complexing compound is protaminewhich is present at about 0.15 mg to about 0.5 mg per 3.5 mg ofderivatized protein.
 3. The microcrystal of claim 2 , wherein thedivalent metal cation is zinc, which is present at about 0.3 mole toabout 0.7 mole per mole of derivatized protein.
 4. The microcrystal ofclaim 3 , wherein the hexamer-stabilizing compound is a phenolicpreservative selected from the group consisting of phenol, m-cresol,o-cresol, p-cresol, chlorocresol, methylparaben, and mixtures thereofand is present in sufficient proportions with respect to the derivatizedprotein to facilitate formation of the R6 hexamer conformation.
 5. Themicrocrystal of claim 4 , wherein the derivatized protein is an acylatedprotein selected from the group consisting of acylated insulin andacylated insulin analogs.
 6. The microcrystal of claim 5 , wherein thederivatized protein is a fatty acid-acylated insulin.
 7. Themicrocrystal of claim 6 , wherein the derivatized protein is insulinthat is acylated with a straight-chain, saturated fatty acid.
 8. Themicrocrystal of claim 7 , wherein the derivatized protein is insulinthat is mono-acylated at the LysB29-Nε amino group of insulin.
 9. Themicrocrystal of claim 8 , wherein the derivatized protein is acylatedwith a fatty acid selected from the group consisting of n-hexanoic acid,n-heptanoic acid, n-octanoic acid, n-nonanoic acid, and n-decanoic acid.10. The microcrystal of claim 9 , wherein the derivatized protein isselected from the group consisting of B29-Nε-hexanoyl-human insulin,B29-Nε-octanoyl-human insulin, and B29-Nε-decanoyl-human insulin. 11.The microcrystal of claim 8 , wherein the derivatized protein isacylated with a fatty acid selected from the group consisting ofn-dodecanoic acid, n-tetradecanoic acid, and n-hexadecanoic acid. 12.The microcrystal of claim 7 , wherein the fatty acid-acylated insulin isa di-acylated insulin that is acylated at the LysB29-Nε-amino group andis also acylated at one N-terminal Nα-amino group, and wherein the fattyacid is selected from the group consisting of n-hexanoic acid,n-heptanoic acid, n-octanoic acid, n-nonanoic acid, and n-decanoic acid.13. The microcrystal of claim 6 , wherein the derivatized protein isinsulin that is acylated with a branched-chain, saturated fatty acid.14. The microcrystal of claim 13 , wherein the branched, saturated fattyacid has from three to ten carbon atoms in its longest branch.
 15. Themicrocrystal of claim 5 , wherein the derivatized protein is a fattyacid-acylated insulin analog.
 16. The microcrystal of claim 15 , whereinthe derivatized protein is an insulin analog that is acylated with astraight-chain, saturated fatty acid.
 17. The microcrystal of claim 16 ,wherein the derivatized protein is mono-acylated at the Nε-amino group.18. The microcrystal of claim 17 , wherein the derivatized protein isacylated with a fatty acid selected from the group consisting ofn-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid, andn-decanoic acid.
 19. The microcrystal of claim 18 , wherein thederivatized protein is selected from the group consisting of fattyacid-acylated animal insulins, fatty acid-acylated monomeric insulinanalogs, fatty acid-acylated deletion analogs, and fatty acid-acylatedpI-shifted insulin analogs.
 20. The microcrystal of claim 19 , whereinthe derivatized protein is fatty acid-acylated des(B30)-human insulinanalog, fatty acid-acylated LysB28,ProB29-human insulin analog, or fattyacid-acylated AspB28-human insulin analog.
 21. The microcrystal of claim20 , wherein the derivatized protein is fatty acid-acylateddes(B30)-human insulin analog.
 22. The microcrystal of claim 17 ,wherein the derivatized protein is acylated with a fatty acid selectedfrom the group consisting of n-dodecanoic acid, n-tetradecanoic acid,and n-hexadecanoic acid.
 23. The microcrystal of claim 22 , wherein thederivatized protein is selected from the group consisting of fattyacid-acylated animal insulins, fatty acid-acylated monomeric insulinanalogs, fatty acid-acylated deletion analogs, and fatty acid-acylatedpI-shifted insulin analogs.
 24. The microcrystal of claim 23 , whereinthe derivatized protein is fatty acid-acylated des(B30)-human insulinanalog, fatty acid-acylated LysB28,ProB29-human insulin analog, or fattyacid-acylated AspB28-human insulin analog.
 25. The microcrystal of claim24 , wherein the derivatized protein is fatty acid-acylateddes(B30)-human insulin analog.
 26. The microcrystal of claim 25 ,wherein the derivatized protein is B29-Nε-myristoyl-des(B30)-humaninsulin analog.
 27. The microcrystal of claim 26 , wherein thederivatized protein is B28-Nε-myristoyl-LysB28,ProB29-human insulinanalog.
 28. The microcrystal of claim 15 , wherein the derivatizedprotein is an insulin analog that is acylated with a branched-chain,saturated fatty acid.
 29. The microcrystal of claim 28 , wherein thebranched chain, saturated fatty acid has from three to ten carbon atomsin its longest branch.
 30. The microcrystal of claim 1 , wherein themicrocrystal has rod-like morphology.
 31. The microcrystal of claim 1 ,wherein the microcrystal has irregular morphology.
 32. A suspensionformulation comprising an insoluble phase and a solution phase, whereinthe insoluble phase is comprised of the microcrystal of claim 1 , andthe solution phase is comprised of water.
 33. A suspension formulationcomprising an insoluble phase and a solution phase, wherein theinsoluble phase is comprised of the microcrystal of claim 2 and thesolution phase is comprised of water.
 34. The suspension formulation ofclaim 33 , wherein the solution phase is further comprised of a phenolicpreservative at a concentration of about 0.5 mg per mL to about 6 mg permL of solution, a pharmaceutically acceptable buffer, and an isotonicityagent.
 35. The suspension formulation of claim 34 , wherein the solutionphase is further comprised of insulin, an insulin analog, an acylatedinsulin, or an acylated insulin analog.
 36. The suspension formulationof claim 35 , wherein the solution phase is comprised of insulin. 37.The suspension formulation of claim 35 , wherein the solution phase iscomprised of an insulin analog.
 38. The suspension formulation of claim37 , wherein the insulin analog is a monomeric insulin analog.
 39. Thesuspension formulation of claim 38 , wherein the insulin analog isLysB28,ProB29-human insulin analog.
 40. The suspension formulation ofclaim 32 , wherein the solution phase is further comprised of zinc andprotamine, wherein the ratio of zinc to derivatized protein in thesuspension formulation is from about 5 to about 7 mole of zinc atoms permole of derivatized protein, and the ratio of protamine to derivatizedprotein in the suspension formulation is from about 0.25 mg to about 0.5mg per mg of derivatized protein.
 41. A process for preparing themicrocrystal of claim 1 comprising: a) dissolving a derivatized protein,a hexamer-stabilizing compound, and a divalent metal cation in anaqueous solvent having a pH that will permit the formation of hexamersof the derivatized protein, and b) adding a complexing compound.
 42. Aprocess for preparing the microcrystal of claim 1 comprising: a)dissolving a derivatized protein, a hexamer-stabilizing compound, and adivalent metal cation in an aqueous solvent having a pH that will notpermit the formation of hexamers of the derivatized protein, and b)adjusting the pH to between about 6.8 and about 7.8; and c) adding acomplexing compound.
 43. A method of treating diabetes comprisingadministering the formulation of claim 32 to a patient in need thereofin a quantity sufficient to regulate blood glucose levels in thepatient.
 44. An amorphous precipitate comprising: a) a derivatizedprotein selected from the group consisting of derivatized insulin,derivatized insulin analogs, and derivatized proinsulins; b) acomplexing compound; c) a hexamer-stabilizing compound; and d) adivalent metal cation.
 45. The amorphous precipitate of claim 44 ,wherein the complexing compound is protamine which is present at about0.15 mg to about 0.5 mg per 3.5 mg of derivatized protein.
 46. Theamorphous precipitate of claim 45 , wherein the divalent metal cation iszinc, which is present at about 0.3 mole to about 0.7 mole per mole ofderivatized protein.
 47. The amorphous precipitate of claim 46 , whereinthe hexamer-stabilizing compound is a phenolic preservative selectedfrom the group consisting of phenol, m-cresol, o-cresol, p-cresol,chlorocresol, methylparaben, and mixtures thereof and is present insufficient proportions with respect to the derivatized protein tofacilitate formation of the R6 hexamer conformation.
 48. The amorphousprecipitate of claim 47 , wherein the derivatized protein is an acylatedprotein selected from the group consisting of acylated insulin andacylated insulin analogs.
 49. The amorphous precipitate of claim 48 ,wherein the derivatized protein is a fatty acid-acylated insulin. 50.The amorphous precipitate of claim 49 , wherein the derivatized proteinis insulin that is acylated with a straight-chain, saturated fatty acid.51. The amorphous precipitate of claim 50 , wherein the derivatizedprotein is insulin that is mono-acylated at the LysB29-Nε amino group ofinsulin.
 52. The amorphous precipitate of claim 51 , wherein thederivatized protein is acylated with a fatty acid selected from thegroup consisting of n-hexanoic acid, n-heptanoic acid, n-octanoic acid,n-nonanoic acid, and n-decanoic acid.
 53. The amorphous precipitate ofclaim 52 , wherein the derivatized protein is selected from the groupconsisting of B29-Nε-hexanoyl-human insulin, B29-Nε-octanoyl-humaninsulin, and B29-Nε-decanoyl-human insulin.
 54. The amorphousprecipitate of claim 51 , wherein the derivatized protein is acylatedwith a fatty acid selected from the group consisting of n-dodecanoicacid, n-tetradecanoic acid, and n-hexadecanoic acid.
 55. The amorphousprecipitate of claim 50 , wherein the fatty acid-acylated insulin is adi-acylated insulin that is acylated at the LysB29-Nε-amino group and isalso acylated at one N-terminal Nα-amino group, and wherein the fattyacid is selected from the group consisting of n-hexanoic acid,n-heptanoic acid, n-octanoic acid, n-nonanoic acid, and n-decanoic acid.56. The amorphous precipitate of claim 49 , wherein the derivatizedprotein is insulin that is acylated with a branched-chain, saturatedfatty acid.
 57. The amorphous precipitate of claim 56 , wherein thebranched, saturated fatty acid has from three to ten carbon atoms in itslongest branch.
 58. The amorphous precipitate of claim 48 , wherein thederivatized protein is a fatty acid-acylated insulin analog.
 59. Theamorphous precipitate of claim 58 , wherein the derivatized protein isan insulin analog that is acylated with a straight-chain, saturatedfatty acid.
 60. The amorphous precipitate of claim 59 , wherein thederivatized protein is mono-acylated at the Nε-amino group.
 61. Theamorphous precipitate of claim 60 , wherein the derivatized protein isacylated with a fatty acid selected from the group consisting ofn-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid, andn-decanoic acid.
 62. The amorphous precipitate of claim 61 , wherein thederivatized protein is selected from the group consisting of fattyacid-acylated animal insulins, fatty acid-acylated monomeric insulinanalogs, fatty acid-acylated deletion analogs, and fatty acid-acylatedpI-shifted insulin analogs.
 63. The amorphous precipitate of claim 62 ,wherein the derivatized protein is fatty acid-acylated des(B30)-humaninsulin analog, fatty acid-acylated LysB28,ProB29-human insulin analog,or fatty acid-acylated AspB28-human insulin analog.
 64. The amorphousprecipitate of claim 63 , wherein the derivatized protein is fattyacid-acylated des(B30)-human insulin analog.
 65. The amorphousprecipitate of claim 60 , wherein the derivatized protein is acylatedwith a fatty acid selected from the group consisting of n-dodecanoicacid, n-tetradecanoic acid, and n-hexadecanoic acid.
 66. The amorphousprecipitate of claim 65 , wherein the derivatized protein is selectedfrom the group consisting of fatty acid-acylated animal insulins, fattyacid-acylated monomeric insulin analogs, fatty acid-acylated deletionanalogs, and fatty acid-acylated pI-shifted insulin analogs.
 67. Theamorphous precipitate of claim 66 , wherein the derivatized protein isfatty acid-acylated des(B30)-human insulin analog, fatty acid-acylatedLysB28,ProB29-human insulin analog, or fatty acid-acylated AspB28-humaninsulin analog.
 68. The amorphous precipitate of claim 67 , wherein thederivatized protein is fatty acid-acylated des(B30)-human insulinanalog.
 69. The amorphous precipitate of claim 68 , wherein thederivatized protein is B29-Nε-myristoyl-des(B30)-human insulin analog.70. The amorphous precipitate of claim 69 , wherein the derivatizedprotein is B28-Nε-myristoyl-LysB28,ProB29-human insulin analog.
 71. Theamorphous precipitate of claim 58 , wherein the derivatized protein isan insulin analog that is acylated with a branched-chain, saturatedfatty acid.
 72. The amorphous precipitate of claim 71 , wherein thebranched chain, saturated fatty acid has from three to ten carbon atomsin its longest branch.
 73. A suspension formulation comprising aninsoluble phase and a solution phase, wherein the insoluble phase iscomprised of the amorphous precipitate of claim 44 , and the solutionphase is comprised of water.
 74. A suspension formulation comprising aninsoluble phase and a solution phase, wherein the insoluble phase iscomprised of the amorphous precipitate of claim 45 and the solutionphase is comprised of water.
 75. The suspension formulation of claim 41, wherein the solution phase is further comprised of a phenolicpreservative at a concentration of about 0.5 mg per mL to about 6 mg permL of solution, a pharmaceutically acceptable buffer, and an isotonicityagent.
 76. The suspension formulation of claim 75 , wherein the solutionphase is further comprised of insulin, an insulin analog, an acylatedinsulin, or an acylated insulin analog.
 77. The suspension formulationof claim 76 , wherein the solution phase is comprised of insulin. 78.The suspension formulation of claim 76 , wherein the solution phase iscomprised of an insulin analog.
 79. The suspension formulation of claim78 , wherein the insulin analog is a monomeric insulin analog.
 80. Thesuspension formulation of claim 79 , wherein the insulin analog isLysB28,ProB29-human insulin analog.
 81. The suspension formulation ofclaim 73 , wherein the solution phase is further comprised of zinc andprotamine, wherein the ratio of zinc to derivatized protein in thesuspension formulation is from about 5 to about 7 mole of zinc atoms permole of derivatized protein, and the ratio of protamine to derivatizedprotein in the suspension formulation is from about 0.25 mg to about 0.5mg per mg of derivatized protein.
 82. A process for preparing theamorphous precipitate of claim 45 comprising:
 110. A suspensionformulation comprising an insoluble phase and a solution phase, whereinthe insoluble phase is a pharmaceutically-active microcrystal comprising(a) a derivative protein selected from the group consisting ofderivatized insulin, derivatized insulin analogs, and derivatizedproinsulins; (b) a complexing compound; (c) a hexamer-stabilizingcompound; and (d) a divalent metal cation.
 111. The suspensionformulation of claim 110 wherein the complexing compound is comprised ofprotamine and the solution phase is comprised of water.
 112. Thesuspension formulation of claim 111 wherein the solution phase isfurther comprised of a phenolic preservative at a concentration of about0.5 mg per mL to about 6 mg per mL of solution, a pharmaceuticallyacceptable buffer, and an isotonicity agent.
 113. The suspensionformulation of claim 112 , wherein the solution phase is furthercomprised of insulin, an insulin analog, an acylated insulin, or anacylated insulin analog.
 114. The suspension formulation of claim 113 ,wherein the solution phase is comprised of insulin.
 115. The suspensionformulation of claim 113 wherein the solution phase is comprised of aninsulin analog.
 116. The suspension formulation of claim 115 , whereinthe insulin analog is a monomeric insulin analog.
 117. The suspensionformulation of claim 116 , wherein the insulin analog isLysB28ProB29-human insulin analog.
 118. The suspension formulation ofclaim 110 , wherein the solution phase is further comprised of zinc andprotamine, wherein the ratio of zinc to derivatized protein in thesuspension formulation is from about 5 to about 7 mole of zinc atoms permole of derivatized protein, and the ratio of protamine to derivatizedprotein in the suspension formulation is from about 0.25 mg to about 0.5mg per mg of derivatized protein.
 119. A process for preparing themicrocrystal of claim 110 comprising: a) dissolving a derivatizedprotein, a hexamer-stabilizing compound, and a divalent metal cation inan aqueous solvent having a pH that will permit the formation ofhexamers of the derivatized protein, and b) adding a complexingcompound.
 120. A process for preparing the microcrystal of claim 110comprising: a) dissolving a derivatized protein, a hexamer-stabilizingcompound, and a divalent metal cation in an aqueous solvent having a pHthat will not permit the formation of hexamers of the derivatizedprotein, and b) adjusting the pH to between about 6.8 and about 7.8; andc) adding a complexing compound.
 121. A method of treating diabetescomprising administering the formulation of claim 110 to a patient inneed thereof in a quantity sufficient to regulate blood glucose levelsin the patient.
 122. An amorphous precipitate comprising: a) aderivatized protein selected from the group consisting of derivatizedinsulin, derivatized insulin analogs, and derivatized proinsulins; b) acomplexing compound; c) a hexamer-stabilizing compound; and d) adivalent metal cation.
 123. The amorphous precipitate of claim 122 ,wherein the complexing compound is protamine which is present at about0.15 mg to about 0.5 mg per 3.5 mg of derivatized protein.
 124. Theamorphous precipitate of claim 123 , wherein the divalent metal cationis zinc, which is present at about 0.3 mole to about 0.7 mole per moleof derivatized protein.
 125. The amorphous precipitate of claim 124 ,wherein the hexamer-stabilizing compound is a phenolic preservativeselected from the group consisting of phenol, m-cresol, o-cresol,p-cresol, chlorocresol, methylparaben, and mixtures thereof and ispresent in sufficient proportions with respect to the derivatizedprotein to facilitate formation of the R6 hexamer conformation.
 126. Theamorphous precipitate of claim 125 , wherein the derivatized protein isan acylated protein selected from the group consisting of acylatedinsulin and acylated insulin analogs.
 127. The amorphous precipitate ofclaim 126 , wherein the derivatized protein is a fatty acid-acylatedinsulin.
 128. The amorphous precipitate of claim 127 , wherein thederivatized protein is insulin that is acylated with a straight-chain,saturated fatty acid.
 129. The amorphous precipitate of claim 128 ,wherein the derivatized protein is insulin that is mono-acylated at theLysB29-Nε amino group of insulin.
 130. The amorphous precipitate ofclaim 129 , wherein the derivatized protein is acylated with a fattyacid selected from the group consisting of n-hexanoic acid, n-heptanoicacid, n-octanoic acid, n-nonanoic acid, and n-decanoic acid.
 131. Theamorphous precipitate of claim 130 , wherein the derivatized protein isselected from the group consisting of B29-Nε-hexanoyl-human insulin,B29-Nε-octanoyl-human insulin, and B29-Nε-decanoyl-human insulin. 132.The amorphous precipitate of claim 131 , wherein the derivatized proteinis acylated with a fatty acid selected from the group consisting ofn-dodecanoic acid, n-tetradecanoic acid, and n-hexadecanoic acid. 133.The amorphous precipitate of claim 132 , wherein the fatty acid-acylatedinsulin is a di-acylated insulin that is acylated at the LysB29-Nε-aminogroup and is also acylated at one N-terminal Nε-amino group, and whereinthe fatty acid is selected from the group consisting of n-hexanoic acid,n-heptanoic acid, n-octanoic acid, n-nonanoic acid, and n-decanoic acid.134. The amorphous precipitate of claim 127 , wherein the derivatizedprotein is insulin that is acylated with a branched-chain, saturatedfatty acid.
 135. The amorphous precipitate of claim 134 , wherein thebranched, saturated fatty acid has from three to ten carbon atoms in itslongest branch.
 136. The amorphous precipitate of claim 128 , whereinthe derivatized protein is a fatty acid-acylated insulin analog. 137.The amorphous precipitate of claim 136, wherein the derivatized proteinis an insulin analog that is acylated with a straight-chain, saturatedfatty acid.
 138. The amorphous precipitate of claim 137, wherein thederivatized protein is mono-acylated at the Nε-amino group.
 139. Theamorphous precipitate of claim 138, wherein the derivatized protein isacylated with a fatty acid selected from the group consisting ofn-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid, andn-decanoic acid.
 140. The amorphous precipitate of claim 139, whereinthe derivatized protein is selected from the group consisting of fattyacid-acylated animal insulins, fatty acid-acylated monomeric insulinanalogs, fatty acid-acylated deletion analogs, and fatty acid-acylatedpI-shifted insulin analogs.
 141. The amorphous precipitate of claim 140,wherein the derivatized protein is fatty acid-acylated des(B30)-humaninsulin analog, fatty acid-acylated LysB28,ProB29-human insulin analog,or fatty acid-acylated AspB28-human insulin analog.
 142. The amorphousprecipitate of claim 141, wherein the derivatized protein is fattyacid-acylated des(B30)-human insulin analog.
 143. The amorphousprecipitate of claim 138, wherein the derivatized protein is acylatedwith a fatty acid selected from the group consisting of n-dodecanoicacid, n-tetradecanoic acid, and n-hexadecanoic acid.
 144. The amorphousprecipitate of claim 143, wherein the derivatized protein is selectedfrom the group consisting of fatty acid-acylated animal insulins, fattyacid-acylated monomeric insulin analogs, fatty acid-acylated deletionanalogs, and fatty acid-acylated pI-shifted insulin analogs.
 145. Theamorphous precipitate of claim 144, wherein the derivatized protein isfatty acid-acylated des(B30)-human insulin analog, fatty acid-acylatedLysB28,ProB29-human insulin analog, or fatty acid-acylated AspB28-humaninsulin analog.
 146. The amorphous precipitate of claim 145, wherein thederivatized protein is fatty acid-acylated des(B30)-human insulinanalog.
 147. The amorphous precipitate of claim 146, wherein thederivatized protein is B29-Nε-myristoyl-des(B30)-human insulin analog.148. The amorphous precipitate of claim 147, wherein the derivatizedprotein is B28-Nε-myristoyl-LysB28,ProB29-human insulin analog.
 149. Theamorphous precipitate of claim 136, wherein the derivatized protein isan insulin analog that is acylated with a branched-chain, saturatedfatty acid.
 150. The amorphous precipitate of claim 149, wherein thebranched chain, saturated fatty acid has from three to ten carbon atomsin its longest branch.
 151. A suspension formulation comprising aninsoluble phase and a solution phase, wherein the insoluble phase iscomprised of the amorphous precipitate of claim 122 , and the solutionphase is comprised of water.
 152. A suspension formulation comprising aninsoluble phase and a solution phase, wherein the insoluble phase iscomprised of the amorphous precipitate of claim 123 and the solutionphase is comprised of water.
 153. The suspension formulation of claim118 , wherein the solution phase is further comprised of a phenolicpreservative at a concentration of about 0.5 mg per mL to about 6 mg permL of solution, a pharmaceutically acceptable buffer, and an isotonicityagent.
 154. The suspension formulation of claim 153, wherein thesolution phase is further comprised of insulin, an insulin analog, anacylated insulin, or an acylated insulin analog.
 155. The suspensionformulation of claim 154, wherein the solution phase is comprised ofinsulin.
 156. The suspension formulation of claim 156, wherein thesolution phase is comprised of an insulin analog.
 157. The suspensionformulation of claim 156, wherein the insulin analog is a monomericinsulin analog.
 158. The suspension formulation of claim 157, whereinthe insulin analog is LysB28,ProB29-human insulin analog.
 159. Thesuspension formulation of claim 153, wherein the solution phase isfurther comprised of zinc and protamine, wherein the ratio of zinc toderivatized protein in the suspension formulation is from about 5 toabout 7 mole of zinc atoms per mole of derivatized protein, and theratio of protamine to derivatized protein in the suspension formulationis from about 0.25 mg to about 0.5 mg per mg of derivatized protein.160. A process for preparing the amorphous precipitate of claim 123comprising: a) dissolving a derivatized protein, a hexamer-stabilizingcompound, and a divalent metal cation in an aqueous solvent having a pHthat will permit the formation of hexamers of the derivatized protein,and b) adding a complexing compound.
 161. A process for preparing theamorphous precipitate of claim 123 comprising: a) dissolving aderivatized protein, a hexamer-stabilizing compound, and a divalentmetal cation in an aqueous solvent having a pH that will not permit theformation of hexamers of the derivatized protein, and b) adjusting thepH to between about 6.8 and about 7.8; and c) adding a complexingcompound.
 162. A method of treating diabetes comprising administeringthe formulation of claim 153 to a patient in need thereof in a quantitysufficient to regulate blood glucose levels in the patient.